Development of an organic polymer monolith column for the nano liquid chromatography fast analysis of monoclonal antibody in infusion bags prepared in a hospital pharmacy.

Poly(butyl methacrylate-co-ethylene dimethacrylate) monolith was in situ prepared in a liquid chromatography capillary column with a 75 µm internal diameter. This monolith offered high permeability (5.3 ± 10-14 m2) and good peak capacity (140 for a 15 cm column length at 300 nl/min with a 20 min gradient time). This is exemplified by its separation ability in reversed mode for subunit analysis of monoclonal antibodies after IdeS digestion (middle-up analysis). The potential of this column was also illustrated for the fast analytical control of therapeutic monoclonal antibodies in standardized infusion bags prepared in advance in a pharmacy department. Linearity analysis revealed the column's capability for accurate quantification analysis of the different dose bandings (in mg) of monoclonal antibodies in <2 min. In addition, lifetime analysis data indicated that the column can be highly reproducible and has a long lifetime with stable and low back pressure. The variations observed on the peak shape and area between unstressed (intact) and stressed monoclonal antibodies indicated that our nano liquid chromatographic method was stability indicating. In addition, using a gradient elution mode, the presence of minor components in the infusion bags was visualized.

New liquid chromatography columns for highlighting the interaction of ligand candidates with humic acid.

Humic acid was the main compound in soil and reduced the availability of some organic compounds in soils. In this work, humic acid was immobilized for the first time on a homemade neutravidin poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary column with a 20 µm i.d. for the screening of potential ligands to humic acid and the evaluation of their molecular recognition mechanism. This homemade humic acid column enabling it to work at very low backpressure (0.60 MPa at 20 nl/min flow rate), had a long lifetime, excellent repeatability, and negligible non-specific binding sites. The performance of this affinity humic acid column was demonstrated by the evaluation of recognition assay for a series of known ligands of humic acid (a series of rodenticide molecules) which is the heart of the fragment-based drug design. In addition, this column was used successfully for highlighting the binding mechanism to humic acid of the severe acute respiratory syndrome coronavirus-2-spike protein. As well this new humic acid miniaturized liquid chromatography column developed in this work could be used in the feature for another solute molecule-humic acid binding studies or for a separative mode.

Performance evaluation of the HPLC diode array and evaporative light scattering detection for the implementation of dose-banded gemcitabine infusion bags.

BACKGROUND: Dose banding (DB) was used to optimise the individualisation of patient treatments with gemcitabine (Gem) in order to improve workload planning at the pharmacy of the University Hospital Centre of Besançon (UHCB). A new simple and fast high-performance liquid chromatographic (HPLC) method was also developed for the quantification of Gem without dilution of the infusion bags. METHODS: Individual doses of Gem preparations were retrospectively analysed over a 1-year period to determine the frequency of prepared doses. Using a maximum gap of 7.5% around the doses chosen, the selected Gem standard doses were 1400 mg, 1600 mg, 1800 mg and 2000 mg. Following the DB scheme, the frequency of prescription of standard and individualised Gem doses was analysed over a period of 10 months. The four selected Gem standard doses were aseptically prepared in polyolefin infusion bags. Each series of 20 bags was stocked under refrigerated storage conditions (4°C) for up to 84 days. The quantification of Gem without dilution of the infusion bags was obtained by the development of a HPLC method coupled to a diode array detector (DAD) or an evaporative light scattering detector (ELSD). RESULTS: During the 10-month period following implementation of the DB, 75.6% of the 1266 prescribed doses were covered by the four standardised preparations. The number of different Gem doses was reduced from 183 to 55. Concerning the Gem quantification, both heteroscedasticity and non-linearity were observed with DAD. Using an ELSD, the trueness values were between 98.59% and 101.52% with excellent repeatability values between 0.66% and 1.42%. CONCLUSION: A new HPLC method has been developed for the quantification of Gem without dilution of the infusion bags prepared in advance as a result of a target DB scheme successfully implemented in our pharmacy department.

Development of a new heparan sulfate proteoglycan (HSPG) chromolith LC column to study the pH dependence binding of peptide vaccines to HSPG and role of human serum albumin on its binding.

Heparan sulfate proteoglycan (HSPG) expressed on immune cell surface participate in antitumor T-cell responses generated in the acidic lymph node (LN) microenvironment. In this work, HSPG was immobilized for the first time on a HPLC chromolith support for studying the effect of extra cellular acidosis in LNs on the binding to HSPG of two peptide vaccines (universal cancer peptide UCP2 and UCP4). This home-made HSPG column enabling to work at high flow-rates, was resistance to change in pH, had a long - life time, an excellent repeatability and negligible non-specific binding sites. The performance of this affinity HSPG column was confirmed by the evaluation of recognition assay for a series of known ligand of HSPG. It was shown that at 37 °C, the UCP2 binding to HSPG versus pH described a sigmoidal shape while UCP4 remained relatively constant in the pH range 5.0-7.5 and lower than the one of UCP2. By the use of an HSA HPLC column, it was shown at 37 °C and in acidic conditions a loss of affinity of UCP2 and UCP4 to HSA. It was demonstrated that upon UCP2/HSA binding, the protonation of the histidine residue in the cluster R(arg) Q(Gln) Hist (H) of the UCP2 peptide allowed to expose more favorably than UCP4 its polar and cationic groups to the negative net charge of HSPG on immune cells. Acidic pHs led to the protonation of the UCP2 residue histidine by flipping the His switch to the on position with a concomitant increase in affinity for the negative net charge of HSPG confirming that UCP2 was more immunogenic than UCP4. As well this HSPG chromolith LC column developed in this work could be used in the feature for other protein - HSPG binding studies or for a separative mode.

Safety, Immunogenicity, and 1-Year Efficacy of Universal Cancer Peptide-Based Vaccine in Patients With Refractory Advanced Non-Small-Cell Lung Cancer: A Phase Ib/Phase IIa De-Escalation Study.

PURPOSE: Universal cancer peptide-based vaccine (UCPVax) is a therapeutic vaccine composed of two highly selected helper peptides to induce CD4+ T helper-1 response directed against telomerase. This phase Ib/IIa trial was designed to test the safety, immunogenicity, and efficacy of a three-dose schedule in patients with metastatic non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with refractory NSCLC were assigned to receive three vaccination doses of UCPVax (0.25 mg, 0.5 mg, and 1 mg) using a Bayesian-based phase Ib followed by phase IIa de-escalating design. The primary end points were dose-limiting toxicity and immune response after three first doses of vaccine. Secondary end points were overall survival (OS) and progression-free survival at 1 year. RESULTS: A total of 59 patients received UCPVax; 95% had three prior lines of systemic therapy. No dose-limiting toxicity was observed in 15 patients treated in phase Ib. The maximum tolerated dose was 1 mg. Fifty-one patients were eligible for phase IIa. The third and sixth dose of UCPVax induced specific CD4+ T helper 1 response in 56% and 87.2% of patients, respectively, with no difference between three dose levels. Twenty-one (39%) patients achieved disease control (stable disease, n = 20; complete response, n = 1). The 1-year OS was 34.1% (95% CI, 23.1 to 50.4), and the median OS was 9.7 months, with no significant difference between dose levels. The 1-year progression-free survival and the median OS were 17.2% (95% CI, 7.8 to 38.3) and 11.6 months (95% CI, 9.7 to 16.7) in immune responders (P = .015) and 4.5% (95% CI, 0.7 to 30.8) and 5.6 months (95% CI, 2.5 to 10) in nonresponders (P = .005), respectively. CONCLUSION: UCPVax was highly immunogenic and safe and provide interesting 1-year OS rate in heavily pretreated advanced NSCLC.

ACE2 and SARS-CoV-2-Main Protease Capillary Columns for Affinity Chromatography: Testimony of the Binding of Dexamethasone and its Carbon Nanotube Nanovector.

In this paper, each of the two following proteins, the angiotensin-converting enzyme 2 (ACE2) and the Main protease (Main pro) of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) were grafted for the first time on homemade neutravidin poly(GMA-co-EDMA) capillary columns for the research of their ligands. The effect of the column diameter on the quantity of immobilized biotinylated protein was studied. For a capillary length of 40 mm, when its internal diameter varied from 75 to 25 µm, the grafted quantity of ACE2 decreased by 85% (from 1.50 to 0.24 µg). Among all the studied ligands, a particular vigilance has been given for dexamethasone, a widely used molecule today for adult patients hospitalized with SARS-CoV-2. Competition experiments were performed with SARS-CoV-2 Receptor Binding Domain used as reference molecule with the ACE2 affinity column to assess the orthosteric binding site of dexamethasone (Dex) on ACE2. This ligand was then immobilized on Multiwall Carbon Nanotubes (Dex/MWCNT). By comparison of the normalized breakthrough curves measured for Dex and Dex/MWCNT on both the ACE2 and Main pro affinity columns, it was showed for the first time that nanovectorisation of Dex with MWCNT enhanced and stabilized its binding to both ACE2 and Main pro. This last result reinforced the use of Dex and the interest of MWCNT for boosting immune health against COVID 19.

Development of nano Bio LC columns for the search of acetylcholinesterase molecular targets.

A novel acetylcholinesterase Nano liquid chromatography capillary column (75 µm i.d. × 50 mm length) was developed for the fast screening of acetylcholinesterase inhibitors and the evaluation of their molecular recognition mechanism. Biotinylated acetylcholinesterase was immobilized on a streptavidin Nano liquid chromatography capillary column. Because of the very strong streptavidin-biotin interaction, the acetylcholinesterase immobilization step performed by frontal analysis is very fast (required less than 10 min), and the amount of immobilized acetylcholinesterase was in the microgram range (1 µg). The yellow anion obtained from the enzymatic reaction detected at 412 nm was achieved within 60 s, and the immobilized acetylcholinesterase retained 96% of the initial activity beyond 90 days. This column was successfully applied for the discrimination of weak affinity ligands to acetylcholinesterase from nonbinders, which is the heart of fragment-based drug design. This column was used for the determination of the IC50 values of a series of inhibitor molecules. In addition, it was demonstrated that competitive experiments could be performed with our miniaturized system to confirm the existence and binding pocket of a ligand to acetylcholinesterase contained in a methanol plant extract. The results revealed that our acetylcholinesterase Nano liquid chromatography capillary column developed in this work represents a useful tool for the rapid screening of inhibitor candidates and evaluation of the action mechanism.

Immobilization of the SARS-CoV-2-receptor binding domain onto methacrylate-based monoliths for nano LC at 30 nL min-1 and application for research of its ligands.

For the design of novel potent molecules against therapeutic protein targets produced in a low quantity or that are very expensive, the development of miniaturized analytical techniques is of crucial importance. One challenging target is the receptor binding domain (RBD) of the SARS-CoV-2-spike protein (S), which mediates the binding of the virus to host cells. In the present study, the RBD protein was thus immobilized on polymethacrylate monoliths prepared in a miniaturized capillary column (25 µm internal diameter; 70 mm length) by in situ polymerization, which could offer low backpressure in Nano LC at 30 nL min-1. The immobilized quantity of the expensive RBD protein on the organic monolith was very low, in the submicrogram range, i.e., 0.060 µg. The immobilization method reduced non-selective interactions between the ligand and the organic monolith matrix and maintained the functionality of RBD due to the high activity rate (96%). The performance of this miniaturized affinity capillary column was demonstrated for the rapid evaluation of a recognition assay induced by 1,2,3,4,6-pentagalloyl glucose (PGG), a known ligand of RBD, and by five other molecules. In addition, it was demonstrated that competitive experiments could be performed with our miniaturized system to reveal the existence of only one type of binding site for three ligands of RBD, namely carbenoxolone, simeprevir and irinotecan. All these results showed the potential of our analytical miniaturized affinity system for the determination of interactions between potential ligands and immobilized RBD of SARS-CoV-2 to aid in the battle against COVID-19.

Development of nano affinity columns for the study of ligand (including SARS-CoV-2 related proteins) binding to heparan sulfate proteoglycans.

The interactions of heparan sulfate proteoglycans (HSPGs) present on the cell surface with target proteins lead to cell signaling and they are considered as viral receptors. The analysis of the recognition mechanism between HSPG and its potential ligands and high-throughput screening in drug discovery thus remain important challenges. Glycidyl methacrylate-based monoliths were thus prepared in situ in miniaturized capillary columns (internal diameter 75 µm) and HSPG was grafted onto them by the use of the Schiff base method. The quantity of grafted HSPG was in the nanogram range (11 nanograms per cm of capillary length). This is of significant importance when working with less available or expensive biological material. Other advantages of our miniaturized capillary column are as follows: (i) the immobilization process of HSPG onto the organic monolithic support was reliable and reproducible. (ii) The resultant affinity capillary column showed a strong resistance to changes in temperature and pH and a negligible non-specific interaction. So as to confirm the proper functioning of our miniaturized capillary column, the molecular recognition by HSPG of five selected compounds including three ligands of interest related to SARS-CoV-2 was studied.

Development of a new nano arginase HPLC capillary column for the fast screening of arginase inhibitors and evaluation of their binding affinity.

A simple and rapid Nano LC method has been developed for the screening of arginase inhibitors. The method is based on the immobilization of biotinylated arginase on a neutravidin functionalized nano HPLC capillary column. The arginase immobilization step performed by frontal analysis is very fast and only takes a few minutes. The miniaturized capillary column of 170 nL (length 5 cm, internal diameter 75 µm) significantly decreased the required amount of used enzyme (25 pmol). This was of significance importance when working with less available or expensive purified enzyme. Non-selective adsorption of the organic monolith matrix was reduced (<6%) and the arginase efficient yield was high (92%). The resultant affinity capillary columns showed excellent repeatability and long lifetime. The arginase reaction product was achieved within 60 s and the immobilized arginase retained 97% of the initial activity beyond 90 days. This novel approach can thus be used for the fast evaluation of recognition assay induced bya series of inhibitor molecules (caffeic acid phenylamide, chlorogenic acid, piceatannol, nor-NOHA acetate) and plant extracts.

Compliance with Early Long-Term Prophylaxis Guidelines for Severe Hemophilia A.

OBJECTIVES: To evaluate the applicability and compliance with guidelines for early initiation of long-term prophylaxis in infants with severe hemophilia A and to identify factors associated with guideline compliance. STUDY DESIGN: This real-world, prospective, multicenter, population-based FranceCoag study included almost all French boys with severe hemophilia A, born between 2000 and 2009 (ie, after guideline implementation). RESULTS: We included 333 boys in the study cohort. The cumulative incidence of long-term prophylaxis use was 61.2% at 3 years of age vs 9.5% in a historical cohort of 39 boys born in 1996 (ie, before guideline implementation). The guidelines were not applicable in 23.1% of patients due to an early intracranial bleeding or inhibitor development. Long-term prophylaxis was delayed in 10.8% of patients. In the multivariate analysis, 2 variables were significantly associated with "timely long-term prophylaxis" as compared with "delayed long-term prophylaxis": hemophilia treating center location in the southern regions of France (OR 23.6, 95% CI 1.9-286.7, P = .013 vs Paris area) and older age at long-term prophylaxis indication (OR 7.2 for each additional year, 95% CI 1.2-43.2, P = .031). Long-term prophylaxis anticipation was observed in 39.0% of patients. Earlier birth year (OR 0.5, 95% CI 0.3-0.8, P = .010 for birth years 2005-2009 vs 2000-2004) and age at first factor replacement (OR 1.9 for each additional year, 95% CI 1.2-3.0, P = .005) were significantly associated with "long-term prophylaxis guideline compliance" vs "long-term prophylaxis anticipation." CONCLUSIONS: This study suggests that long-term prophylaxis guidelines are associated with increased long-term prophylaxis use. However, early initiation of long-term prophylaxis remains a challenge.

No effect of tDCS of the primary motor cortex on isometric exercise performance or perceived fatigue.

Anodal transcranial direct current stimulation (tDCS) of the primary motor cortex has been reported to improve isometric exercise performance without changing corticospinal excitability. One possible cause for this may be the previous use of relatively high (2 mA) current intensities, which have inconsistent effects on corticospinal excitability. The present pre-registered study aimed to replicate previously reported ergogenic effects of 2 mA tDCS and examine whether 1 mA anodal tDCS both improved isometric exercise performance and perceived fatigue, and more reliably altered corticospinal excitability. On three separate occasions, participants performed a sustained submaximal isometric knee extension until failure after receiving either 1, 2 mA or sham anodal tDCS. Corticospinal excitability of the knee extensors was measured using transcranial magnetic stimulation immediately before and after tDCS. Rating of fatigue was recorded throughout the isometric exercise. Neither 1 nor 2 mA tDCS improved exercise performance, or reduced perceived fatigue, compared with sham stimulation. There was also no effect of tDCS on the corticospinal excitability of the knee extensors. We found no effect of tDCS on either exercise performance, perceived fatigue or corticospinal excitability. This study adds to the growing body of literature reporting no ergogenic effect of tDCS. Large pre-registered replications of previously reported effects are now required before tDCS can be considered an effective method to improve exercise performance.

Harnessing demographic differences in organizations: What moderates the effects of workplace diversity?

To account for the double-edged nature of demographic workplace diversity (i.e,. relational demography, work group diversity, and organizational diversity) effects on social integration, performance, and well-being-related variables, research has moved away from simple main effect approaches and started examining variables that moderate these effects. While there is no shortage of primary studies of the conditions under which diversity leads to positive or negative outcomes, it remains unclear which contingency factors make it work. Using the Categorization-Elaboration Model as our theoretical lens, we review variables moderating the effects of workplace diversity on social integration, performance, and well-being outcomes, focusing on factors that organizations and managers have control over (i.e., strategy, unit design, human resource, leadership, climate/culture, and individual differences). We point out avenues for future research and conclude with practical implications. © 2015 The Authors. Journal of Organizational Behavior published by John Wiley & Sons, Ltd.

Development of a simple technique for the coating of monolithic silica with pristine boron nitride nanotubes (BNNTs): HPLC chromatographic applications.

In this paper, a novel and very simple homogeneous coating of a monolithic silica HPLC support using pristine boron nitride nanotubes (BNNTs) was d0.escribed. The chromatographic support was coated with BNNTs in a non covalent way to preserve the nanotube structure. A solution of BNNTs dispersed in dimethylacetamide (DMAc) was pumped through the column at a flow-rate of 0.3mL/min for 24h at room temperature. Strong interaction between amino groups and the BNNT surfaces induces the adsorption of the BNNTs on the silica, while the stable solvation in DMAc hampers further adsorption of the tubes. The excellent stability of the non covalent BNNT-coating on the monolithic silica in view of application for HPLC was also demonstrated. It was shown that this novel stationary phase was efficient for the HPLC isocratic or gradient mode separation of molecules of different structure such as phenol derivatives, alkylbenzene or doping agents (steroids). As well, this simple technique of BNNT immobilization offers new perspectives for the BNNT-coating on the surfaces of a wide range of solid substrates.

Comparative binding to DR4 and DR5 receptors of TRAIL and BNNTs/PAHE/mPEG-DSPE/TRAIL nanoparticles.

TRAIL is a member of the tumor necrosis factor family of cytokines, which induces apoptosis of cancer cells, thanks to its binding to its cognate receptors DR5 and DR4. We have recently demonstrated that nanovectorization of TRAIL with single-walled carbon nanotubes enhanced TRAIL affinity to DR5. In this paper, 1-pyrenebutyric acid N-hydroxysuccinimide ester functionalized boron nitride nanotubes (BNNTs) were used to anchor the TRAIL protein. The resulting BNNT/1-pyrenebutyric acid N-hydroxysuccinimide ester nanotubes were mixed with methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-conjugates so as to allow a good dispersion of these nanoparticle TRAIL (NPT) in aqueous solution. The difference of binding between NPT and soluble TRAIL to DR4 and DR5 receptors was then studied by the use of affinity chromatography. DR4 and DR5 receptors were thus immobilized on a chromatographic support, and the binding of the 2 ligands TRAIL and NPT to DR4 and DR5 was studied in the temperature range 30°C to 50°C. Negative enthalpy (ΔH) values indicated that van der Waals interactions and hydrogen bonding are engaged favorably at the ligand-receptor interface. It was shown that their rank-ordered affinities were strongly different in the sequence TRAILDR4  < NPTDR4  < TRAILDR5  < NPTDR5 , and the highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on BNNTs. Taken together, our results demonstrated that nanovectorization of TRAIL with BNNTs enhanced its binding to both DR4 and DR5 receptors at 37°C. Our novel nanovector could potentially be used for delivering TRAIL to cells for cancer treatment.

An HPLC method associated with a thermodynamic analysis to compare the binding of TRAIL and its nanovectorized form to death receptors DR4 and DR5 and their relationship to cytotoxicity.

TRAIL is a member of the TNF family of cytokines which induces apoptosis of cancer cells via its binding to its cognate receptors, DR5 a high affinity site and DR4 a site of low affinity. Our working group has recently demonstrated that nanovectorization of TRAIL with single wall carbon nanotubes (abbreviated NPT) enhanced TRAIL affinity to the high affinity site DR5 and increased pro apoptotic potential in different human tumor cell lines. In this paper, the DR4 low affinity site was immobilized on a chromatographic support and the effect of temperature on a wide temperature range 1°C-50°C was studied to calculate the thermodynamic parameters of the binding of TRAIL and NPT to DR4 and DR5 receptors. For the first time the heat capacity changes for the different binding processes were determined. At a physiological pH (7.4) the heat capacity changes for the binding of NPT to DR4 and DR5 were respectively equal to -0.91kJ/molK and -0.28kJ/molK and those obtained for the binding of TRAIL to DR4 and DR5 were respectively equal to -1.54kJ/molK and -1.05kJ/molK. By the use of differential scanning calorimetry (DSC), a phase transition (∼12°C for DR5, ∼4°C for DR4) between a disordered (low temperature) and an ordered (high temperature) solid like state visualized in the receptor structure confirmed the temperature dependence of binding affinity enthalpy ΔH for soluble TRAIL and its nanovectorized form to its cognate receptors. In the low temperature domain, the positive ΔH values contribute non-favourably to the free energy of binding, TRAIL and NPT described similar affinities for DR4 and DR5. For the high temperature domain, negative ΔH values indicated that van der Waals interactions and hydrogen bonding are engaged favourably at the ligand - receptor interface. Above 30°C, their rank-ordered affinities were thus strongly different in the sequence: TRAILDR4

Heparan Sulfate Proteoglycans Promote Telomerase Internalization and MHC Class II Presentation on Dendritic Cells.

Telomerase is a prototype-shared tumor Ag and represents an attractive target for anticancer immunotherapy. We have previously described promiscuous and immunogenic HLA-DR-restricted peptides derived from human telomerase reverse transcriptase (hTERT) and referred as universal cancer peptide (UCP). In nonsmall cell lung cancer, the presence of spontaneous UCP-specific CD4 T cell responses increases the survival of chemotherapy-responding patients. However, the precise mechanisms of hTERT's uptake, processing, and presentation on MHC-II molecules to stimulate CD4 T cells are poorly understood. In this work, by using well-characterized UCP-specific CD4 T cell clones, we showed that hTERT processing and presentation on MHC-II involve both classical endolysosomal and nonclassical cytosolic pathways. Furthermore, to our knowledge, we demonstrated for the first time that hTERT's internalization by dendritic cells requires its interaction with surface heparan sulfate proteoglycans. Altogether, our findings provide a novel mechanism of tumor-specific CD4 T cell activation and will be useful for the development of novel cancer immunotherapies that harness CD4 T cells.

Enhanced DR5 binding capacity of nanovectorized TRAIL compared to its cytotoxic version by affinity chromatography and molecular docking studies.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of cancer cells when bound to its cognate receptors, TRAIL-R1 and TRAIL-R2 (DR4 and DR5), without being toxic to healthy cells. Nanovectorized TRAIL (abbreviated as NPT) is 10 to 20 times more efficient than one of the most potent soluble TRAIL used in preclinical studies (His-TRAIL). To determine whether differences in affinity may account for NPT superiority, a thermodynamic study was undertaken to evaluate NPT versus TRAIL binding affinity to DR5. Docking calculations showed that TRAIL in homotrimer configuration was more stable than in heterotrimer, because of the presence of one Zn ion in its structure. Indeed, TRAIL trimers can have head-to-tail orientations when Zn is missing. Altogether these data suggest that TRAIL homotrimer structures are predominant in solution and then are grafted on NPT. When docked to DR5, NPT carrying TRAIL homotrimer leads to a more stable complex than TRAIL monomer-based NPT. To comfort these observations, the extracellular domain of DR5 was immobilized on a chromatographic support using an "in situ" immobilization technique. The determination of the thermodynamic data (enthalpy ∆H° and entropy ∆S°*) of TRAIL and NPT binding to DR5 showed that the binding mechanism was pH dependent. The affinity of NPT to DR5 increased with pH, and the ionized energy was more important for NPT than for soluble TRAIL. Moreover, because of negative values of ∆H° and ∆S°* quantities, we demonstrated that van der Waals and hydrogen bonds governed the strong NPT-DR5 association for pH > 7.4 (as for TRAIL alone). Copyright © 2016 John Wiley & Sons, Ltd.

An HPLC chromatographic framework to analyze the ß-cyclodextrin/solute complexation mechanism using a carbon nanotube stationary phase.

A carbon nanotube (CNT) stationary phase was used for the first time to study the ß-cyclodextrin (ß-CD) solute complexation mechanism using high performance liquid chromatography (HPLC). For this, the ß-CD was added at various concentrations in the mobile phase and the effect of column temperature was studied on both the retention of a series of aniline and benzoic acid derivatives with the CNT stationary phase and their complexation mechanism with ß-CD. A decrease in the solute retention factor was observed for all the studied molecules without change in the retention order. The apparent formation constant KF of the inclusion complex ß-CD/solute was determined at various temperatures. Our results showed that the interaction of ß-CD with both the mobile phase and the stationary phase interfered in the complex formation. The enthalpy and entropy of the complex formation (ΔHF and ΔSF) between the solute molecule and CD were determined using a thermodynamic approach. Negative enthalpies and entropies indicated that the inclusion process of the studied molecule in the CD cavity was enthalpically driven and that the hydrogen bonds between carboxylic or aniline groups and the functional groups on the ß-CD rim play an important role in the complex formation.

H2O2: a Ca(2+) or Mg(2+)-sensing function in statin passive diffusion.

In a previous paper Guillaume's group demonstrated that magnesium (Mg(2+) concentration range 0.00-2.60 mm) increased the passive diffusion of statins and thus played a role in their potential toxicity. In order to confirm an increase in this passive diffusion by divalent salt cations, the role of calcium chloride (CaCl2) on the statin-immobilized artificial membrane (IAM) association was studied. It was demonstrated that calcium supplementation (Ca(2+) concentration range 0.00-3.25 mm) increases the statin passive diffusion. In addition, it was shown that the Ca(2+) effect on the statin-IAM association is higher than that of Mg(2+). These results show that Ca(2+) enhances the passive diffusion of drugs into biological membranes and thus their potential toxicity. Also, addition of H2O2 to the medium showed a hyperbolic response for the statin passive diffusion and this effect was enhanced for the highest Ca(2+) or Mg(2+) concentrations in the medium. H2O2 is likely to interact with the polar head groups of the IAM through dipole-dipole interactions. The conformational changes in H2O2-IAM result in a higher degree of exposure of hydrophobic areas, thus explaining why the binding of pravastatin, which showed the lowest logP value, was less affected by H2O2. This result shows the significant contribution of H2O2 and thus the oxidative stress on the statin passive diffusion. Much of the sensitivity derives from the action of Ca(2+) or Mg(2+), in turn supported the idea that H2O2 may serve a Ca(2+) or Mg(2+) sensing function in statin passive diffusion.