The Complementary Developing Solvent technique, originally developed for high-performance thin-layer chromatography, employs solvent systems with distinct polarity ranges to achieve enhanced chromatographic resolution while covering a broad chemical space. This study demonstrates the successful adaptation of low polarity developing solvent and high polarity developing solvent systems to normal-phase flash chromatography for systematic fractionation. The system suitability test used in high-performance thin layer chromatography, namely the Universal HPTLC Mix, enabled direct comparisons between high-performance thin layer chromatography and flash chromatography, demonstrating a conserved polarity-driven retention hierarchy across chromatographic scales. To confirm compound identities and elution order, each chromatographic peak was collected and analyzed by tandem mass spectrometry, with MS/MS spectra manually matched against reference standards. Quantitative chromatographic performance was benchmarked using effective peak capacity (Pc), yielding a value of 16.41 for the high-polarity developing system, indicative of a globally balanced separation. In addition, a systematic isocratic step fractionation strategy was implemented by segmenting each elution gradient (low polarity developing solvent and high polarity developing solvent) into seven predefined polarity windows. This strategy was applied on a crude methanolic extract of Sideritis scardica Griseb., confirming robust and reproducible fractionation boundaries in a complex botanical matrix. Collecting a single fraction per isocratic step significantly reduces the number of samples for subsequent analysis such as bioassays and untargeted metabolomic profiling, thereby accelerating natural product discovery and dereplication workflows.

Myotonic dystrophy type 1 (OMIM #160900) is a multisystemic, autosomal, and dominantly inherited pathology. It is characterized by an expansion (>50) of trinucleotides [CTG] n on the 3' untranslated region of the dystrophia myotonica protein kinase gene, transcribed into RNA [CUG] n , aggregating in the nucleus as foci. The most accepted pathological mechanism considers the sequestration and dysregulation of proteins, including splicing factors (muscle-blind-like splicing regulator 1 and CUG-binding protein 1) by pathological RNA. Different therapeutic strategies to overcome this defect exist, such as the use of small molecules targeting the pathological RNA to prevent sequestration. Among these small molecules, pentamidine (PTMD), an antiprotozoal drug, has previously been shown to interact with a [CUG] n repeat. In this context, we developed a fine-tuned affinity capillary electrophoresis (ACE) method that provides highly repeatable migration times. This is a crucial point, as all subsequent calculations for apparent affinity constant (Kaapp ) determination rely on them. Afterward, we quantified the interactions between pentamidine and two RNA probes: [CUG]95, representative of the disease, and [CUG]14 as a negative control. The results indicated an excellent affinity between [CUG]95 and PTMD. Selectivity was assessed by comparing the Kaapp values: (6.1 ± 0.4) × 103 M-1 and (3.8 ± 0.3) × 103 M-1 for the positive and negative controls, respectively. For the first time, an orthogonal UPLC-UV methodology was applied to corroborate the ACE results. No significant differences were observed between the two analytical methods. To overcome the documented toxicity related to pentamidine, different libraries of small molecules have been investigated by the ACE method. Among these, two compounds, neomycin and chloroquine, demonstrated interactions with the pathological RNA model. Therefore, their affinities toward the positive and negative controls were quantified to assess the selectivity of these potential therapeutic candidates.

Despite advancements in treating metastatic melanoma, many patients exhibit resistance to targeted therapies. Our study focuses on ATP1A1, a sodium pump subunit associated with cancer development. We aimed to assess ATP1A1 prognostic value in melanoma patients and examine the impact of its ligand, bufalin, on melanoma cell lines in vitro and in vivo. High ATP1A1 expression (IHC) correlated with reduced overall survival in melanoma patients. Resistance to BRAF inhibitor was linked to elevated ATP1A1 levels in patient biopsies (IHC, qPCR) and cell lines (Western blot, qPCR). Additionally, high ATP1A1 mRNA expression positively correlated with differentiation/pigmentation markers based on data from The Cancer Genome Atlas (TCGA) databases and Verfaillie proliferative gene signature analysis. Bufalin specifically targeted ATP1A1 in caveolae (, proximity ligation assay) and influenced Src phosphorylation (Western blot), thereby disrupting multiple signaling pathways (phosphokinase array). In vitro, bufalin induced apoptosis in melanoma cell lines by acting on ATP1A1 (siRNA experiments) and, in vivo, significantly impeded melanoma growth using a nude mouse xenograft model with continuous bufalin delivery via an osmotic pump. In conclusion, our study demonstrates that ATP1A1 could serve as a prognostic marker for patient survival and a predictive marker for response to BRAF inhibitor therapy. By targeting ATP1A1, bufalin inhibited cell proliferation, induced apoptosis in vitro, and effectively suppressed tumor development in mice. Thus, our findings strongly support ATP1A1 as a promising therapeutic target, with bufalin as a potential agent to disrupt its tumor-promoting activity.

The synthesis of dialkyl carbonates, versatile compounds with applications in organic synthesis, pharmaceuticals, and polymers, has attracted considerable attention due to their environmentally benign nature. Here, we describe the selective bimolecular nucleophilic substitution (SN2) N 2) reaction between primary and secondary alkyl iodides with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-based carbon dioxide-binding organic liquids. We show that TBD is a great candidate for bulk carbon dioxide and alcohol binding at 100 degrees C. degrees C. TBDbased carbonate salts are selective for SN2 N 2 processes, allowing them to work with highly reactive alkyl iodide while eliminating unwanted base quaternization either in acetonitrile or in bulk at both 21 degrees C degrees C and 65 degrees C. degrees C. The high reactivity of these TBD-based carbon dioxide-binding organic liquids toward backside SN2 N 2 processes at low temperature is explained by the presence of the TBD.H+ + guanidinium, revealing a unique metal-free cation-assisted SN2 N 2 ion-pair process.

The synthesis of dialkyl carbonates, versatile compounds with applications in organic synthesis, pharmaceuticals, and polymers, has attracted considerable attention due to their environmentally benign nature. Here, we describe the selective bimolecular nucleophilic substitution (SN2) reaction between primary and secondary alkyl iodides with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-based carbon dioxide-binding organic liquids. We show that TBD is a great candidate for bulk carbon dioxide and alcohol binding at 100°C. TBD-based carbonate salts are selective for SN2 processes, allowing them to work with highly reactive alkyl iodide while eliminating unwanted base quaternization either in acetonitrile or in bulk at both 21°C and 65°C. The high reactivity of these TBD-based carbon dioxide-binding organic liquids toward backside SN2 processes at low temperature is explained by the presence of the TBD.H+ guanidinium, revealing a unique metal-free cation-assisted SN2 ion-pair process.

Malaria remains to date one of the most devastating parasitic diseases worldwide. The fight against this disease is rendered more difficult by the emergence and spread of drug-resistant strains. The need for new therapeutic candidates is now greater than ever. In this study, we investigated the antiplasmodial potential of toad venoms. The wide array of bioactive compounds present in Bufonidae venoms has allowed researchers to consider many potential therapeutic applications, especially for cancers and infectious diseases. We focused on small molecules, namely bufadienolides, found in the venom of Rhinella marina (L.). The developed bio-guided fractionation process includes a four solvent-system extraction followed by fractionation using flash chromatography. Sub-fractions were obtained through preparative TLC. All samples were characterized using chromatographic and spectrometric techniques and then underwent testing on in vitro Plasmodium falciparum cultures. Two strains were considered: 3D7 (chloroquine-sensitive) and W2 (chloroquine-resistant). This strategy highlighted a promising activity for one compound named resibufogenin. With IC50 values of (29 ± 8) μg/mL and (23 ± 1) μg/mL for 3D7 and W2 respectively, this makes it an interesting candidate for further investigation. A molecular modelling approach proposed a potential binding mode of resibufogenin to Plasmodium falciparum adenine-triphosphate 4 pump as antimalarial drug target.

The rise and spread of resistant Plasmodium falciparum strains are responsible for an increase in therapeutic failures in many of the regions endemic with malaria. The need for new therapeutic candidates is now more urgent than ever. Animal venoms have long been considered as interesting resources to exploit in terms of potential therapeutic candidates. Among these, the cutaneous secretions of toads constitute a rich and diverse source of bioactive molecules. We focused on two different species: Bufo bufo and Incilius alvarius. The dried secretions underwent a solvent-based extraction and were submitted to a systematic bio-guided fractionation approach using preparative thin-layer chromatography. Initial crude extracts were tested in vitro for their antiplasmodial activity. Based on these results, only crude extracts displaying IC50 < 100 μg/mL were considered for further fractionation. All extracts and fractions, including those that did not display antiplasmodial properties, were characterized by chromatographic (LC-UV/MS) and spectrometric techniques (HRMS). Antiplasmodial activity was evaluated in vitro using a chloroquine-sensitive strain (3D7) and a resistant one (W2). Toxicity was assessed on normal human cells for the samples displaying IC50 < 100 μg/mL. Crude extracts from Bufo bufo secretions exhibited no appreciable antiplasmodial activities. However, the methanol and dichloromethane extracts from Incilius alvarius secretions gave IC50 of (34 ± 4) μg/mL and (50 ± 1) μg/mL respectively when tested on W2 strain. No significant effect was observed on 3D7. This poison would warrant further investigation in terms of its antiplasmodial potential. Following preliminary characterization, it was revealed that the fractions of interest contained mainly bufotoxins, bufagins and alkaloids.

The reduction of K2 Cr2 O7 solutions by H2 O2 was studied by nuclear magnetic resonance (NMR) relaxometry and UV-vis spectroscopy in HCl/KCl buffer (pH 2), NaCl/glycine/HCl buffer (pH 3), and sodium acetate/acetic acid buffer (pH 4). Because of Cr(III) paramagnetism, 1/T1 and 1/T2 of the solutions increase during the reduction of diamagnetic Cr(VI). This increase is proportional to the produced Cr(III) concentration. Using different initial H2 O2 concentrations, partially reduced Cr(VI) samples were prepared and studied by T1 and T2 relaxometry and by UV-vis spectroscopy. The correlation between the relaxation rates and the concentration of Cr(VI) remaining in the sample, measured by spectroscopy, was excellent. It was possible, thanks to the measurement of T2 , to study the kinetics of the reduction of K2 Cr2 O7 by H2 O2 in the pH 3 and pH 4 buffers. The reduction of Cr(VI) by ascorbic acid was successfully monitored by NMR relaxometry in the pH 2 buffer. The presence of complexing molecules/ions was shown to drastically influence the nuclear magnetic relaxation dispersion profiles of reduced K2 Cr2 O7 solutions: Both relaxation rates are divided by ~5 when citrate or acetate ions are present and by ~3 in the presence of ascorbic acid. Therefore, the comparison of relaxation results obtained in different reaction mixtures must be done carefully. When all the solutions are set to pH 0, which prevents any complexation, the longitudinal and transverse relaxation rates of all samples become comparable. Finally, as a proof of concept for a turbid solution, the kinetics of the reduction of a K2 Cr2 O7 solution by aluminum powder in the pH 2 buffer was successfully monitored.

Biosensing using optical fibers allows the detection of low concentrations of analytes. To exploit this property, we study a cost-effective fiber-based solution to target the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a biomarker for malaria diagnosis. In this work, unclad multimode optical fiber probes (400 µm core diameter) are coated with a thin gold film to excite Surface Plasmon Resonance (SPR), yielding high sensitivity to surrounding medium and more precisely to target-bioreceptor interactions. They are connected to a portable white light source and a spectrometer for read-out. The covalent immobilization of anti-PfHRP2 antibodies on gold and its passivation allowed specific detection in different types of media. The use of secondary antibodies as amplifiers in a sandwich assay also allowed us to improve the sensitivity of the method. Different blocking agents (fish gelatin, bovine serum albumin and casein) were studied to optimize the surface selectivity. The detection of PfHRP2 was first calibrated using spiked proteins into phosphate buffer saline (PBS) and then tested in vitro using fresh cultures of Plasmodium falciparum. The shifts observed with our technique were also compared with results obtained from an ELISA (gold-standard technique for detection and quantification of targets), and commercial paper-based lateral-flow assays, usually used on field. As malaria affects many people worldwide, improvement and multiplexing of this technique can be a perspective to overcome the limitations of currently available point-of-care tests in the future.

The reduction of K2 Cr2 O7 solutions by H2 O2 was studied by NMR relaxometry and UV-Vis spectroscopy in HCl/KCl buffer (pH2), NaCl/Glycine/HCl buffer (pH3) and sodium acetate/acetic acid buffer (pH4). Because of Cr (III) paramagnetism, 1/T1 and 1/T2 of the solutions increase during the reduction of diamagnetic Cr (VI). This increase is proportional to the produced Cr (III) concentration. Using different initial H2 O2 concentrations, partially reduced Cr (VI) samples were prepared and studied by T1 and T2 relaxometry and by UV-Vis spectroscopy. The correlation between the relaxation rates and the concentration of Cr (VI) remaining in the sample, measured by spectroscopy, was excellent. It was possible, thanks to the measurement of T2 , to study the kinetics of the reduction of K2 Cr2 O7 by H2 O2 in the pH3 and pH4 buffers. The reduction of Cr (VI) by ascorbic acid was successfully monitored by NMR relaxometry in the pH2 buffer. The presence of complexing molecules/ions was shown to drastically influence the Nuclear Magnetic Relaxation Dispersion profiles of reduced K2 Cr2 O7 solutions: both relaxation rates are divided by ~5 when citrate or acetate ions are present and by ~3 in the presence of ascorbic acid. Therefore, the comparison of relaxation results obtained in different reaction mixtures must be done carefully. When all the solutions are set to pH0, which prevents any complexation, the longitudinal and transverse relaxation rates of all samples become comparable. Finally, as a proof of concept for a turbid solution, the kinetics of the reduction of a K2 Cr2 O7 solution by aluminum powder in the pH2 buffer was successfully monitored.

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.

Malaria is a parasitic disease, endemic in many tropical and sub-tropical countries. Malaria is a well-known disease, familiar to almost all people in endemic regions, as they or their family are regularly confronted with it; everyone in these regions has probably experienced the disease, at least once in their life. To investigate the social perceptions of malaria in Burkina Faso, including its diagnosis-driven treatment, we have conducted a survey in both urban (Saint Camille Hospital, Ouagadougou HOSCO) and rural (Boussé Hospital) areas. Fifty-six individuals, mostly representatives of the society variability, were surveyed by questionnaires and 2 focus groups were organized with traditional healers. In general, populations seem to have grasped the causes, symptoms and means of preventing the disease. However, the majority of interviewees make a marked confusion between malaria and dengue; dengue fever is considered like a severe form of malaria. The care modalities (modern and/or traditional medicine) are plural and the choice of therapeutic practice depends on both the socio-economic conditions and education level of the patient. Whereas some patients mark preferences for one type of medicine, others simultaneously recourse to both; for these, a medicine does not outperform the other and their combination multiplies the chances of a quick recovery. Whether for modern or traditional medicine, the diagnosis is considered very important for effective disease management. Modern medicine uses diagnostic tools based on light microscopy and immunochromatography (rapid diagnostic tests; RDT); traditional medicine has its own diagnostic logic but nevertheless recognizes modern medicine diagnosis to guide its therapy. 90 % of those interviewed first use modern medicine to seek an accurate diagnosis of their disease and thus to receive adequate treatment. Presumptive treatments are still widely prescribed and accepted by most patients who trust the judgment of their caregiver, not perceiving any benefit to an objective diagnosis. In front of a negative diagnosis, patient reactions are diverse, some accepting investigations for other diseases (45 %), others opting for self-medication (15 %), others resorting to traditional medicine (20 %). All are unanimous in the importance of diagnosis and are in favor of in-development diagnostic technologies, provided these obviously meet the features of reliability, ease of use, availability and, of course, economical accessibility.

Background: Malaria remains the first global parasitic endemic disease with more than 400,000 deaths per year; there is a definite need for prevention measures and treatments, but also for rapid and low-cost diagnostic methods. Hemozoin, a detoxification polymer formed from heme by the parasite, and a likely biomarker of infection, has enticed many studies aiming at its whole blood determination. But, to our best knowledge, no accurate, precise and sensitive analytical method has been developed that could be implemented in endemic regions. Objectives: Our group recently proposed a macroscopic trapping-dissolution method based on the paramagnetic properties of hemozoin crystals. The present paper further develops the concept into a workable fluidic device, validating an instrumental method that could be applied to the diagnosis of malaria. Results: In the newly developed integrated on-line system, the paramagnetic crystals are successively trapped through a superparamagnetic microbeads gradient field, dissolved by an alkaline solution, losing magnetic properties, eluted and quantified by spectrophotometry at 405 nm. The analysis time is comprised between 10 and 15 min. The performances of the method have been evaluated both on aqueous suspensions of beta-hematin (a synthetic pigment with physical and paramagnetic properties analogous to those of hemozoin) and Plasmodium cultures, including the response function, linearity, precision, trueness, accuracy and quantification limits. From beta-hematin suspensions and Plasmodium falciparum 3D7 cultures, the limit of detection would correspond to 80 and 55 parasites/mu L whole blood, respectively (0.05 and 0.033 mu g hemozoin/mL). Conclusion: In the absence of a reference method for the determination of hemozoin, its value as a malaria biomarker remains a matter of heavy debates. The newly developed magneto-chromatographic on-line system can discriminate the presence and absence of hemozoin in a sample but also accurately and precisely determine its level; application to whole-blood samples from strictly graded patients will allow to precise the usefulness of hemozoin for malaria diagnosis and/or prognosis.

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.

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 (K a) 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.

For the first time, the effectiveness of triazolinedione (TAD) click chemistry onto aliphatic polycarbonates (APC) is demonstrated. Statistic copolymers carrying click-reactive conjugated diene (in a ratio of 10%) are synthesized via organocatalyzed ring-opening polymerization. The highly efficient click reaction of TADs carrying simple butyl and phenyl functions are confirmed by 1 H-NMR and DSC. Network formation using a bivalent TAD is also performed and simply characterized by DSC. This post-polymerization functionalization of biocompatible and biodegradable APC pave the way to easy and versatile "on-demand" materials design.

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.

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the al-isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d(3)-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O:MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm x 3 mm; 3 pm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.