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Background Early malaria diagnosis and its profiling require the development of new sensing platforms enabling rapid and early analysis of parasites in blood or saliva, aside the widespread rapid diagnostic tests (RDTs). Methods This study shows the performance of a cost-effective optical fiber-based solution to target the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Unclad multimode optical fiber probes are coated with a thin gold film to excite Surface Plasmon Resonance (SPR) yielding high sensitivity to bio-interactions between targets and bioreceptors grafted on the metal surface. Results Their performances are presented in laboratory conditions using PBS spiked with growing concentrations of purified target proteins and within in vitro cultures. Two probe configurations are studied through label-free detection and amplification using secondary antibodies to show the possibility to lower the intrisic limit of detection. Conclusions As malaria hits millions of people worldwide, the improvement and multiplexing of this optical fiber technique can be of great interest, especially for a future purpose of using multiple receptors on the fiber surface or several coated-nanoparticles as amplifiers.

Dystrophia myotonica type 1 (DM1) results from nuclear sequestration of splicing factors by a messenger RNA (mRNA) harboring a large (CUG)n repeat array transcribed from the causal (CTG)n DNA amplification. Several compounds were previously shown to bind the (CUG)n RNA and release the splicing factors. We now investigated for the first time the interaction of an aliphatic polycarbonate carrying guanidinium functions to DM1 DNA/RNA model probes by affinity capillary electrophoresis. The apparent association constants (Ka) were in the range described for reference compounds such as pentamidine. Further macromolecular engineering could improve association specificity. The polymer presented no toxicity in cell culture at concentrations of 1.6–100.0 μg/mL as evaluated both by MTT and real-time monitoring xCELLigence method. These promising results may lay the foundation for a new branch of potential therapeutic agents for DM1.

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na+ /K+ -ATPase pump by inhibiting the cardiotonic steroid dependent-site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt-sensitive hypertensive states such as essential hypertension, congestive heart-failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin-layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid-phase extraction coupled with fractionation on high-performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid-phase extraction combined with high-performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time-consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.

The understanding of nucleic acids-ligand (proteins, nucleic acids or various xenobiotics) interactions is of fundamental value, representing the basis of complex mechanisms that govern life. The development of improved therapeutic strategies, as well as the much expected breakthroughs in case of currently untreatable diseases often relies on the elucidation of such biomolecular interactions. Capillary electrophoresis (CE) is becoming an indispensable analytical tool in this field of study due to its high versatility, ease of method development, high separation efficiency, but most importantly due to its low sample and buffer volume requirements. Most often the availability of the compounds of interest is severely limited either by the complexity of the purification procedures or by the cost of their synthesis. Several reviews covering the investigation of protein-protein and protein-xenobiotics interactions by CE have been published in the recent literature; however none of them promotes the use of these techniques in the study of nucleic acid interactions. Therefore, various CE techniques applicable for such interaction studies are discussed in detail in the present review. The paper points out the particular features of these techniques with respect the estimation of the binding parameters, in analytical signal acquisition and data processing, as well as their current shortcomings and limitations.

Melanoma is the most common cancer in young adults, with a constantly increasing incidence. Metastatic melanoma is a very aggressive cancer with a 5-year survival rate of about 22−25%. This is, in most cases, due to a lack of therapies which are effective on the long term. Hence, it is crucial to find new therapeutic agents to increase patient survival. Toad venoms are a rich source of potentially pharmaceutically active compounds and studies have highlighted their possible effect on cancer cells. We focused on the venoms of two different toad species: Bufo bufo and Rhinella marina. We screened the venom crude extracts, the fractions from crude extracts and isolated biomolecules by studying their antiproliferative properties on melanoma cells aiming to determine the compound or the combination of compounds with the highest antiproliferative effect. Our results indicated strong antiproliferative capacities of toad venoms on melanoma cells. We found that these effects were mainly due to bufadienolides that are cardiotonic steroids potentially acting on the Na+/K+ ATPase pump which is overexpressed in melanoma. Finally, our results indicated that bufalin alone was the most interesting compound among the isolated bufadienolides because it had the highest antiproliferative activity on melanoma cells., SCOPUS: ar.j, info:eu-repo/semantics/published

Unfortunately the name of Jean Jacques Vanden Eynde was missing as co-author of this contribution. The correct list of authors is: Ioan O. Neaga, Stephanie Hambye, Ede Bodoki, Claudio Palmieri, Jean Jacques Vanden Eynde, Eugenie Ansseau, Alexandra Belayew, Radu Oprean, Bertrand Blankert.

Myotonic dystrophy type 1 (DM1) is an autosomal dominantly inherited degenerative disease with a slow progression. At the present, there is no commercially available treatment, but sustained effort is currently undertaken for the development of a promising lead compound. In the present paper we report the development of a fast, versatile, and cost-effective affinity capillary electrophoresis (ACE) method for the screening and identification of potential drug candidates targeting pathological ARN probes relevant for DM1. The affinity studies were conducted in physiologically relevant conditions using 50 mM HEPES buffer (pH 7.4) in a fused silica capillary dynamically coated with poly(ethylene oxide), by testing a library of potential ligands against (CUG)50 RNA as target probe with a total run time of 4–5 h/ligand. For the most promising ligands, their affinity parameters were assessed and some results formerly reported on the affinity of pentamidine (PTMD) and neomycin against CUG repeats were confirmed. To the best of the authors’ knowledge, the estimated binding stoichiometry for some of the tested compounds (i.e., ~ 121:1 for PTMD against the tested RNA probe) is reported for the first time. Additionally, the potential of a novel pentamidine like compound, namely 1,2-ethane bis-1-amino-4-benzamidine (EBAB) with much lower in vivo toxicity than its parent compound has also been confirmed studying its effect on a live cell model by fluorescence microscopy. Further tests, such as the evaluation of the rescue in the mis-splicing of the involved genes, can be performed to corroborate the potential therapeutic value of EBAB in DM1 treatment.

The research for new in vitro screening tools for predictive metabolic profiling of drug candidates is of major interest in the pharmaceutical field. The main motivation is to avoid late rejection in drug development and to deliver safer drugs to the market. Thanks to the superparamagnetic properties of iron oxide nanoparticles, a flow bioreactor has been developed which is able to perform xenobiotic metabolism studies. The selected cell line (HepaRG) maintained its metabolic competencies once iron oxide nanoparticles were internalized. Based on magnetically trapped cells in a homemade immobilization chamber, through which a flow of circulating phase was injected to transport nutrients and/or the studied xenobiotic, off-line and online (when coupled to a high-performance liquid chromatography chain) metabolic assays were developed using diclofenac as a reference compound. The diclofenac demonstrated a similar metabolization profile chromatogram, both with the newly developed setup and with the control situation. Highly versatile, this pioneering and innovative instrumental design paves the way for a new approach in predictive metabolism studies.

Pentamidine isethionate (PTMD) is an antiprotozoal agent used in different parasitic diseases as Human African Trypanosomiasis or Pneumocystis pneumonia. Given its side effects, numerous analogs are still under development worldwide. PTMD has been recently described having a potential activity in myotonic dystrophy (type 1). Here we present an UPLC method coupled to fluo or PDA detection for PTMD and one analog determination in rat plasma or urine. The chromatographic separation was achieved on a Acquity UPLC® HSS T3 analytical column using a mobile phase combining formic acid 0.1% (v/v) and acetonitrile (ACN) at a constant flow rate of 0.4 mL/min. Preliminary, an innovative μSPE (solid phase extraction) procedure using Oasis® WCX sorbent was processed and gave satisfying and reproducible results in terms of extraction yields. Additionally, the methods were successfully validated using the accuracy profiles approach (β=95% and acceptance limits=15%) over the ranges 2.88-287.52 ng/mL and from 143.76 ng/mL to 1.72 μg/mL in rat plasma and urine for PTMD and for EBAB, from 4.23 to 423.39 ng/mL and from 211.69 ng/mL to 2.54 μg/mL for plasma and urine, respectively. The validated protocols were applied to a pharmacokinetic (PK) study on rats and permitted to point out some relevant PK parameters on PTMD and its studied analog.

Three novel LC-UV methods for the determination of pentamidine (PTMD) and two of its new analogs in rat plasma are described. The chromatographic conditions (wavelength, acetonitrile percentage in the mobile phase, internal standard) were optimized to have an efficient selectivity. A pre-step of extraction was simultaneously developed for each compound. For PTMD, a solid phase extraction (SPE) with Oasis® HLB cartridges was selected, while for the analogs we used protein precipitation with acetonitrile. SPE for PTMD gave excellent results in terms of extraction yield (99.7 ± 2.8) whereas the recoveries for the analogs were not so high but were reproducible as well (64.6 ± 2.6 and 36.8 ± 1.6 for analog 1 and 2, respectively). By means of a recent strategy based on accuracy profiles (β-expectation tolerance interval), the methods were successfully validated. β was fixed at 95% and the acceptability limits at ±15% as recommended by the FDA. The method was successfully validated for PTMD (29.6-586.54 ng/mL), analog 1 (74.23-742.3 ng/mL) and analog 2 (178.12-890.6 ng/mL). The first concentration level tested was considered as the LLOQ (lower limit of quantification) for PTMD and analog 1 whereas for analog 2, the LLOQ was not the first level tested and was raised to 178.12 ng/mL. © 2010 Elsevier B.V. All rights reserved.