Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Screening for pancreatic lipase natural modulators by capillary electrophoresis hyphenated to spectrophotometric and conductometric dual detection.

The search for novel pancreatic lipase (PL) inhibitors has gained increasing attention in recent years. For the first time, a dual detection capillary electrophoresis (CE)-based homogeneous lipase assay was developed employing both the offline and online reaction modes. The hydrolysis of 4-nitrophenyl butyrate (4-NPB) catalyzed by PL into 4-nitrophenol and butyrate was monitored by spectrophotometric and conductimetric detection, respectively. The assays presented several advantages such as economy in consumption (few tens of nanoliters for online assays to few tens of microliters for offline assays), no modification of lipase, rapidity (<10 min) and versatility. Tris/MOPS (10 mM, pH 6.6) was used as the background electrolyte and the incubation buffer for enzymatic reactions. We confirmed that in the conditions of the study (small substrate 4-NPB, 37 °C, pH 6.6), the PL was active even in the absence of dipalmitoylphosphatidylcholine (DPPC) vesicles, generally used to mimic the lipid-water interface. This was confirmed by the maximum velocity (Vmax) and the Michaelis-Menten constant (Km) values that were the same order of magnitude in the absence and presence of DPPC. The developed method was used to screen crude aqueous plant extracts and purified compounds. We were able to identify the promising PL inhibition of hawthorn leaf herbal infusions at 1 mg mL-1 (37%) and PL activation by fresh and dry hawthorn flowers (∼24%). Additionally, two triterpenoids purified from extracts of oakwood were identified for the first time as potent PL inhibitors demonstrating 51 and 58% inhibition at 1 mg mL-1, respectively.

Investigation of lipase-ligand interactions in porcine pancreatic extracts by microscale thermophoresis.

The evaluation of binding affinities between large biomolecules and small ligands is challenging and requires highly sensitive techniques. Microscale thermophoresis (MST) is an emerging biophysical technique used to overcome this limitation. This work describes the first MST binding method to evaluate binding affinities of small ligands to lipases from crude porcine pancreatic extracts. The conditions of the MST assay were thoroughly optimized to successfully evaluate the dissociation constant (Kd) between pancreatic lipases (PL) and triterpenoid compounds purified from oakwood. More precisely, the fluorescent labeling of PL (PL*) using RED-NHS dye was achieved via a buffer exchange procedure. The MST buffer was composed of 20 mM NaH2PO4 + 77 mM NaCl (pH 6.6) with 0.05% Triton-X added to efficiently prevent protein aggregation and adsorption, even when using only standard, uncoated MST capillaries. Storage at -20 °C ensured stability of PL* and its fluorescent signal. MST results showed that crude pancreatic extracts were suitable as a source of PL for the evaluation of binding affinities of small ligands. Quercotriterpenoside-I (QTT-I) demonstrated high PL* binding affinity (31 nM) followed by 3-O-galloylbarrinic acid (3-GBA) (500 nM) and bartogenic acid (BA) (1327 nM). To enrich the 50 kDa lipase responsible for the majority of hydrolysis activity in the crude pancreatic extracts, ammonium sulfate precipitation was attempted and its efficiency confirmed using capillary electrophoresis (CE)-based activity assays and HRMS. Moreover, to accurately explain enzyme modulation mechanism, it is imperative to complement binding assays with catalytic activity ones.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Polyethylene glycol crowding effect on hyaluronidase activity monitored by capillary electrophoresis.

To mimic the activity of hyaluronidase in natural environment, the hydrolysis of hyaluronic acid (HA) by hyaluronidase was investigated for the first time in the presence of crowding agents using capillary electrophoresis (CE) as a simple and reliable technique for conducting enzymatic assay. Polyethylene glycol (PEG) 6000 was selected as a model crowder and the hyaluronic acid degradation catalyzed by bovine testes hyaluronidase (BTH) was carried out at different PEG concentrations (0%, 10%, and 17%). After optimization of the CE analytical method and enzymatic assay, the degradation products were monitored at different HA concentrations. At 10% of PEG and 0.3 mg mL-1 of HA, the activity of the enzyme was significantly reduced showing inconvenient interactions of PEG with the hyaluronidase blocking the release of hydrolysis products. A similar reduction of hyaluronidase activity was observed at 1 mg mL-1 of HA due to the presumable formation of the BTH-substrate complex. The experimental curves obtained by CE also evidence that the overall kinetics are governed by the hydrolysis of hexasaccharide intermediates. Finally, the effect of PEG on hyaluronidase activity was evaluated in the presence of natural or synthetic inhibitors. Our results show a significant difference of the inhibitors' affinity toward hyaluronidase in the presence of PEG. Surprisingly, the presence of the crowding agent results in a loss of the inhibition effect of small polycyclic inhibitors, while larger charged inhibitors were less affected. In this work, CE analyses confirm the importance of mimicking the cellular environment for the discovery and development of reliable inhibitors. Graphical abstract.

Screening and Evaluation of Dermo-Cosmetic Activities of the Invasive Plant Species Polygonum cuspidatum.

Polygonum cuspidatum (P. cuspidatum) is among the world's most problematic invasive plant species with negative ecological, socio-economic and security consequences. Management operations in areas invaded systematically generate a large quantity of plant waste, most often without outlets. Using this plant material could constitute a new alternative treatment for sustainable management. P. cuspidatum is well known to have numerous biological properties, containing notably stilbenes, quinones, flavonoids and phenolic acids. The present work proposes a reliable strategy using powerful techniques for the screening and the evaluation of the dermo-cosmetic potential of its aerial parts (AP) and root parts (RP). To the best of our knowledge, only antioxidant and anti-tyrosinase activities were previously evaluated on P. cuspidatum among the targets studied (superoxide dismutase, hyaluronidase, elastase, collagenase and tyrosinase). The results revealed strong antioxidant and anti-collagenase activities, moderate anti-hyaluronidase activity, while weak anti-elastase and anti-tyrosinase activities were observed for ethanolic extracts. Different standards selected and screened on the same targets made it possible to correlate the observed residual activities of produced extracts of P. cuspidatum from Savoie Mont Blanc and their chemical compositions. A structure-activity study was thus conducted on main molecular families, widely represented in the genus Polygonum.

Kinetic theory of hyaluronan cleavage by bovine testicular hyaluronidase in standard and crowded environments.

In this paper, we introduce a comprehensive kinetic model describing the enzymatic cleavage of hyaluronan (HA) by bovine testicular hyaluronidase (BTH). Our theory focuses specifically on the late stage of the hydrolysis, where the concentrations of a limited number of oligomers may be determined experimentally with accuracy as functions of time. The present model was applied to fit different experimental sets of kinetic data collected by capillary electrophoresis at two HA concentrations and three concentrations of PEG crowder (0, 10, 17% w/w). Our theory seems to apply universally, irrespective of HA concentration and crowding conditions, reproducing to an excellent extent the time evolution of the individual molar fractions of oligomers. Remarkably, we found that the reaction mechanism in the late degradation stage essentially reduces to the cleavage or transfer of active dimers. While the recombination of dimers is the fastest reaction, the rate-limiting step turns out to be invariably the hydrolysis of hexamers. Crowding, HA itself or other inert, volume-excluding agents, clearly boosts recombination events and concomitantly slows down all fragmentation pathways. Overall, our results bring a novel and comprehensive quantitative insight into the complex reaction mechanism underlying enzymatic HA degradation. Importantly, rationalizing the effect of crowding not only brings the intricate conditions of in-vivo settings a little closer, but also emerges as a powerful tool to help pinpointing relevant kinetic pathways in complex systems.

Water-Based Extraction of Bioactive Principles from Blackcurrant Leaves and Chrysanthellum americanum: A Comparative Study.

The water-based extraction of bioactive components from flavonoid-rich medicinal plants is a key step that should be better investigated. This is especially true when dealing with easy-to-use home-made conditions of extractions, which are known to be a bottleneck in the course for a better control and optimization of the daily uptake of active components from medicinal plants. In this work, the water-based extraction of Blackcurrant (Ribes nigrum) leaves (BC) and Chrysanthellum americanum (CA), known to have complementary pharmacological properties, was studied and compared with a previous work performed on the extraction of Hawthorn (Crataegus, HAW). Various extraction modes in water (infusion, percolation, maceration, ultrasounds, microwaves) were compared for the extraction of bioactive principles contained in BC and CA in terms of extraction yield, of amount of flavonoids, phenolic compounds, and proanthocyanidin oligomers, and of UHPLC profiles of the extracted compounds. The qualitative and quantitative aspects of the extraction, in addition to the kinetic of extraction, were studied. The optimized easy-to-use-at-home extraction protocol developed for HAW was found very efficient to easily extract bioactive components from BC and CA plants. UHPLC-ESI-MS and high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were also implemented to get more qualitative information on the specific and common chemical compositions of the three plants (including HAW). Their antihyaluronidase, antioxidant, and antihypertensive activities were also determined and compared, demonstrating similar activities as the reference compound for some of these plants.