Impact Of Experimental Conditions Research Articles

Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts.

Chronic kidney disease (CKD) patients undergoing dialysis are at high risk of bone fractures. CKD-induced mineral and bone disorder is extended to periodontal disease due to changes in the ionic composition of saliva in CKD patients, dysregulating mineralization, hindering regeneration and thereby promoting the progression of dental complications. Despite the importance of cementum for overall oral health, the mechanisms that regulate its development and regeneration are not well comprehended, and a lack of sufficient in vitro experimental models has hindered research progress. In this study, the impact of experimental conditions on the calcification of cementoblasts was systematically investigated, aimed at establishing a standardized and validated model for the calcification of cementoblasts. The effects of phosphate, calcium, ascorbic acid, β-glycerolphosphate, dexamethasone, and fetal calf serum on the calcification process of cementoblasts were analyzed over a wide range of concentrations and time points by investigating calcium content, cell viability, gene expression and kinase activity. Cementoblasts calcified in a concentration- and time-dependent manner with higher concentrations of supplements cause a higher degree of calcification but decreased cell viability. Phosphate and calcium have a significantly stronger effect on cementoblast calcification processes compared to osteogenic supplements: ascorbic acid, β-glycerolphosphate, and dexamethasone induce calcification over a wide range of osteogenic signalling pathways, with osteopontin being a central target of gene regulation. Conversely, treatment with ascorbic acid, β-glycerolphosphate, and dexamethasone leads to activating only selected pathways, especially promoting bone sialoprotein expression. The developed and validated cementoblast calcification protocol, incubating up to 60% confluent cementoblasts with 1.9 mmol L-1 of phosphate supplementation for a reasonable, multi-pathway calcification induction and 10 mmol L-1 β-glycerolphosphate, 75 µmol L-1 ascorbic acid and 10 nmol L-1 dexamethasone for a reasonable osteogenic differentiation-based calcification induction, provides standard in vitro experimental models for better understanding cementoblast function and regeneration.

The Impact of Experimental Conditions on Cell Mechanics as Measured with Nanoindentation.

The evaluation of cell elasticity is becoming increasingly significant, since it is now known that it impacts physiological mechanisms, such as stem cell differentiation and embryogenesis, as well as pathological processes, such as cancer invasiveness and endothelial senescence. However, the results of single-cell mechanical measurements vary considerably, not only due to systematic instrumental errors but also due to the dynamic and non-homogenous nature of the sample. In this work, relying on Chiaro nanoindenter (Optics11Life), we characterized in depth the nanoindentation experimental procedure, in order to highlight whether and how experimental conditions could affect measurements of living cell stiffness. We demonstrated that the procedure can be quite insensitive to technical replicates and that several biological conditions, such as cell confluency, starvation and passage, significantly impact the results. Experiments should be designed to maximally avoid inhomogeneous scenarios to avoid divergences in the measured phenotype.

'The impact of experimental conditions on cell mechanics as 2 measured with nanoindentation' (Stiffness Data)

'The impact of experimental conditions on cell mechanics as 2 measured with nanoindentation' (Stiffness Data)

Immobilization of per- and polyfluoroalkyl substances (PFAS): Comparison of leaching behavior by three different leaching tests

The evaluation of PFAS immobilization performance in laboratory experiments, especially the long-term stability, is a challenge. To contribute to the development of adequate experimental procedures, the impact of experimental conditions on the leaching behavior was studied. Three experiments on different scales were compared: batch, saturated column, and variably saturated laboratory lysimeter experiments. The Infinite Sink (IS) test – a batch test with repeated sampling – was applied for PFAS for the first time. Soil from an agricultural field amended with paper-fiber biosolids polluted with various perfluoroalkyl acids (PFAAs; 655 μg/kg ∑18PFAAs) and polyfluorinated precursors (1.4 mg/kg ∑18precursors) was used as the primary material (N-1). Two types of PFAS immobilization agents were tested: treatment with activated carbon-based additives (soil mixtures: R-1 and R-2), and solidification with cement and bentonite (R-3). In all experiments, a chain-length dependent immobilization efficacy is observed. In R-3, the leaching of short-chain PFAAs was enhanced relative to N-1. In column and lysimeter experiments with R-1 and R-2, delayed breakthrough of short-chain PFAAs (C4) occurred (> 90 days; in column experiments at liquid-to-solid ratio (LS) > 30 L/kg) with similar temporal leaching rates suggesting that leaching in these cases was a kinetically controlled process. Observed differences between column and lysimeter experiments may be attributed to varying saturation conditions. In IS experiments, PFAS desorption from N-1, R-1, and R-2 is higher than in the column experiments (N-1: +44 %; R-1: +280 %; R-2: +162 %), desorption of short-chain PFAS occurred predominantly in the initial phase (< 14 days). Our findings demonstrate that sufficient operating times are essential in percolation experiments, e.g., in column experiments >100 days and LS > 30 L/kg. IS experiments may provide a faster estimate for nonpermanent immobilization. The comparison of experimental data from various experiments is beneficial to evaluate PFAS immobilization and to interpret leaching characteristics.

Impact of Experimental Conditions on Extracellular Vesicles' Proteome: A Comparative Study.

Extracellular vesicle (EV) research is a rapidly developing field, mainly due to the key role of EVs in intercellular communication and pathophysiological processes. However, the heterogeneity of EVs challenges their exploration and the establishment of gold-standard methods. Here, we aimed to reveal the influence of technical changes on EV biology and the reliability of experimental data. We used B16F1 melanoma cells as a model and applied nanoparticle tracking analysis, mass spectrometry (LC-MS/MS) and pathway enrichment analysis to analyze the quantity, size distribution, proteome and function of their small EVs (sEVs) produced in sEV-depleted fetal bovine serum (FBS)-containing medium or serum-free medium. Additionally, we investigated the effects of minor technical variances on the quality of sEV preparations. We found that storage of the isolates at -80 °C has no adverse effect on LC-MS/MS analysis, and an additional washing step after differential ultracentrifugation has a minor influence on the sEV proteome. In contrast, FBS starvation affects the production and proteome of sEVs; moreover, these vesicles may have a greater impact on protein metabolism, but a smaller impact on cell adhesion and membrane raft assembly, than the control sEVs. As we demonstrated that FBS starvation has a strong influence on sEV biology, applying serum-free conditions might be considered in in vitro sEV studies.

Long-Term Pterostilbene Supplementation of a High-Fat Diet Increases Adiponectin Expression in the Subcutaneous White Adipose Tissue

Pterostilbene (Pt) is a natural phenol found in blueberries and grapes; it shows remarkable biomedical activities similar to those of resveratrol, but its higher bioavailability is a major advantage for possible biomedical applications. Our group has recently demonstrated that long-term (30 weeks) administration of Pt to mice maintained on a high-fat diet counters weight gain and promotes browning of subcutaneous white adipose tissue (sWAT). By Real-time quantitative PCR and Western Blot analysis of the sWAT and visceral white adipose tissue (vWAT) from the same mice used in the previous study, we show here that Pt induced a long-term increase of Adiponectin, Interleukin 10 and of M2 macrophage marker Cd206. The effects were observed in sWAT, while no significant changes were detected in vWAT. The process taking place seems to mimic that occurring in sWAT during cold-induced browning. Analysis of a few pro-inflammatory cytokines (Interleukin 6, Tumor necrosis factor α) and of the NFkB pathway did not reveal marked effects of Pt supplementation. In summary, the mechanisms and processes through which Pt acts in adipose tissue appear to closely mimic those set in motion by cold-induced browning, and point to a possible impact of experimental conditions in the final output of a nutraceutical intervention.

Optimization of the Transwell® System for Assessing the Dissolution Behavior of Orally Inhaled Drug Products through In Vitro and In Silico Approaches.

The aim of this study was to further evaluate and optimize the Transwell® system for assessing the dissolution behavior of orally inhaled drug products (OIDPs), using fluticasone propionate as a model drug. Sample preparation involved the collection of a relevant inhalable dose fraction through an anatomical mouth/throat model, resulting in a more uniform presentation of drug particles during the subsequent dissolution test. The method differed from previously published procedures by (1) using a 0.4 µm polycarbonate (PC) membrane, (2) stirring the receptor compartment, and (3) placing the drug-containing side of the filter paper face downwards, towards the PC membrane. A model developed in silico, paired with the results of in vitro studies, suggested that a dissolution medium providing a solubility of about 5 µg/mL would be a good starting point for the method’s development, resulting in mean transfer times that were about 10 times longer than those of a solution. Furthermore, the model suggested that larger donor/receptor and sampling volumes (3, 3.3 and 2 mL, respectively) will significantly reduce the so-called “mass effect”. The outcomes of this study shed further light on the impact of experimental conditions on the complex interplay of dissolution and diffusion within a volume-limited system, under non-sink conditions.

Zinc chloride modification of sage leaves powder and its application as an adsorbent for KMnO4 removal from aqueous solutions

Sage Leaves Powder was modified by 30% w/w of Zink Chloride (ZnCl2) and used as a cheap adsorbent for potassium permanganate (KMnO4) adsorption from aqueous media. The characterization of the synthesized adsorbent showed diversity in its functional groups at 1030, 1700, and 2930 cm−1. The impact of experimental conditions on the adsorption was investigated. It was found that these experimental conditions except pH have positive effects on the efficiency of this adsorbent. The initial KMnO4 concentration of 1500 mg l−1, the temperature of 60 °C and the solution pH of 1.5 were found to be the ideal conditions in this adsorption. The equilibrium data were analyzed using three isotherm models. Langmuir model was found to be the best model applied in this work. The adsorption capacities were 714.29, 769.23, 833.33 and 909.09 (mg g−1) at 30, 40, 50 and 60 (°C), respectively. The parameters of the adsorption kinetics were evaluated by three kinetic models. The kinetic results confirm that the 2nd order described well the experimental data. Thermodynamic outcomes suggest that this adsorption is a spontaneous and endothermic process. The obtained results make from this adsorbent a good option for KMnO4 removal from wastewater in terms of its low-cost and adsorption performance.

On the Centrality Determination with Forward Proton Detectors

The forward proton detectors, already installed at the Large Hadron Collider, are studied in the context of heavy-ion collisions. The potential of such detectors in measuring the nuclear debris coming from the spectator fragments is presented. The geometric acceptance of the forward proton detectors for different debris is estimated. The impact of experimental conditions and the Fermi motion on the acceptance is studied. A possibility of the collision impact parameter reconstruction from the measurement of nuclear fragments is discussed.

Magnetic Resonance Elastography of Rodent Brain.

Magnetic resonance elastography (MRE) is a non-invasive imaging technique, using the propagation of mechanical waves as a probe to palpate biological tissues. It consists in three main steps: production of shear waves within the tissue; encoding subsequent tissue displacement in magnetic resonance images; and extraction of mechanical parameters based on dedicated reconstruction methods. These three steps require an acoustic-frequency mechanical actuator, magnetic resonance imaging acquisition, and a post-processing tool for which no turnkey technology is available. The aim of the present review is to outline the state of the art of reported set-ups to investigate rodent brain mechanical properties. The impact of experimental conditions in dimensioning the set-up (wavelength and amplitude of the propagated wave, spatial resolution, and signal-to-noise ratio of the acquisition) on the accuracy and precision of the extracted parameters is discussed, as well as the influence of different imaging sequences, scanners, electromagnetic coils, and reconstruction algorithms. Finally, the performance of MRE in demonstrating viscoelastic differences between structures constituting the physiological rodent brain, and the changes in brain parameters under pathological conditions, are summarized. The recently established link between biomechanical properties of the brain as obtained on MRE and structural factors assessed by histology is also studied. This review intends to give an accessible outline on how to conduct an elastography experiment, and on the potential of the technique in providing valuable information for neuroscientists.

Bayesian inference method for in situ thermal conductivity and heat capacity identification: Comparison to ISO standard

Non-destructive thermal diagnosis is necessary for identification and quantification of structural defects and verification of construction performances. In this paper, we test different inverse heat transfer models to identify the thermal conductivity of a building’s wall in its environment. The approaches proposed and used in this paper rely only on non-intrusive measurements: inside and outside wall surface temperatures and heat flow through the wall. First, a Bayesian statistical dynamic inference method, which has the advantage to quantify the unknown parameter and its credible interval, is presented. This method considers the uncertainties of the measured temperature and heat flow data and of the unknown thermal properties. Markov chain Monte Carlo (MCMC) algorithm is used to explore the posterior distribution. Then, the average and the dynamic procedures ISO 9869 (I. 9869-1, 2014) are introduced. Finally, the probabilistic distributions of the unknown parameters are presented and compared to the standard results. The impact of experimental conditions (average indoor-to-outdoor temperature) and the measurements length on the accuracy of the results are discussed. The relationship between the number of iterations of the MCMC, time series length, shape of the prior distribution and accuracy are studied as well as the simulation time to run the inverse models. The Bayesian approach gives the most accurate results and has the advantage of considering several unknowns (conductivity and volumetric heat capacity), which is not the case for the studied standard. The Bayesian method needs much shorter time series than the ISO standard and produces robust results at all times of year, including when the average indoor-to-outdoor temperature difference was low.

Drug combinations and impact of experimental conditions on relative recovery in in vitro microdialysis investigations

The need for pharmacokinetic knowledge about antibiotics directly at the site of infection, typically the interstitial space fluid (ISF) of tissues, is gaining acceptance for effective and safe treatment. One option to acquire such data is the microdialysis technique employing a catheter with a semipermeable membrane inserted directly in the ISF. A prerequisite is catheter calibration, e.g. via retrodialysis, yielding a conversion factor from measured to true ISF concentrations, termed relative recovery. This value can be influenced by various factors. The present investigation assessed the impact of three of them on relative recovery using seven drugs: (I) drug combinations/order, (II) air in the microdialysis system, (III) flow rate changes inherent when using common in vivo microdialysis pumps.All experiments were performed in a standardised in vitro microdialysis system. (I) Relative recovery of single antibiotics (linezolid, meropenem, cefazolin, metronidazole, tigecycline) was determined in microdialysis and retrodialysis settings and compared with values using either antibiotic or antibiotic+analgesic (acetaminophen and metamizole) combinations or single drugs with reversed microdialysis order. For assessing these factors for lower relative recovery values (as in in vivo), these were mimicked by increasing the flow rate for linezolid. (II) For the impact of air, linezolid relative recovery of freshly carbonated solutions was compared to degassed ones in microdialysis and retrodialysis settings. For each condition in (I) and (II), summary statistics of relative recovery were calculated and for the impact of the factors a linear mixed-effect model developed. (III) From samples taken during an automatic flush sequence (15 μL/min) of an in vivo pump and afterwards switching to the flow rate of 1 and 2 μL/min for 120 min, the time necessary for relative recovery to reach equilibrium was determined.(I) High relative recovery values (flow rate 2 μL/min: ≥84%; flow rate 5 μL/min: ≥65%) were observed for all investigated single drugs. Intra- and intercatheter variability ranged from 0.3%–11% and 3%–25%, respectively. Based on these values and on the statistical model, the impact of drug combination versus single drug as well as of reversed order was small with changes in relative recovery of smaller equal 9%. (II) Compared to degassed solutions, relative recovery in carbonated solutions was 23% and 19% lower (relative reduction) in the microdialysis and retrodialysis setting, respectively, with increased intercatheter variability (up to 37%). (III) As expected, relative recovery increased after the flush sequence and was constant 10–15 min after the switch to the typical 1 and 2 μL/min flow rate.Given the intercatheter variability, combinations and the order of drugs showed minor but clinically negligible impact on relative recovery. In contrast, air in the microdialysis catheter/system caused falsely low and inconsistent relative recovery values and must be avoided when performing a trial. Also changes in flow rate at the end of pump flush sequence impacted relative recovery. Hence, a sufficient equilibration time of 10–15 min prior to sampling should be implemented in sampling protocols. In vitro microdialysis presents a highly valuable complementary platform to clinical microdialysis studies impacting the design, sampling schedule and data analysis of such trials to gain knowledge of target-site pharmacokinetics for contributing to better informed decisions in the individual patient/special populations in future.

Agonist stimulation at human μ opioid receptors in a [35S]GTPγS incorporation assay: observation of “bell-shaped” concentration–response relationships under conditions of strong receptor G protein coupling

Context: The appearance of “bell”- (or “inverted U”-) shaped agonist concentration–response curves (CRCs) in in vitro pharmacological experiments is a frequently observed but poorly communicated phenomenon. In the context of G protein coupled receptor research, it is commonly attributed to the recruitment of secondary targets or to desensitization or feedback processes, but the concrete background of these observations often remains intriguing. Objective: Here, we addressed the subject of bell-shaped agonist CRCs at the µ opioid receptor (µOR) by testing the impact of experimental conditions favoring G protein coupling. Methods: G protein activation by recombinant human µORs heterologously expressed in CHO cells was assessed in [35S]GTPγS binding assays using the opioid ligands DAMGO, morphine, fentanyl and naloxone. Experimental conditions were varied by changing the NaCl (10–300 mM) and the GDP concentration (0.3–30 µM). Results: Both the sodium and the GDP concentration were inversely related to G protein coupling, as evident by an increase in basal [35S]GTPγS incorporation at low sodium and low GDP levels and by the concomitant appearance of the partial agonist activity of the µOR antagonist, naloxone. Bell-shaped CRCs were observed for the efficacious agonists DAMGO, fentanyl and morphine, and this phenomenon was promoted by low sodium as well as by low GDP concentrations. Conclusion: µOR agonist CRCs show a non-monotonic behavior with a decline of maximal stimulation under conditions of strong receptor-G protein coupling, and this behavior is visible at the level of G protein activation itself.

Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions

Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy. PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the molecular mechanism of PARP1 activation and its functional consequences after mustard treatment in general. Such a study is presented in an accompanying article (Mangerich et al., 2016).

Phobos: A novel software for recording rodents’ behavior during the thigmotaxis and the elevated plus-maze test

Evaluation of fear and anxiety levels offers valuable insight on the impact of experimental conditions. The elevated plus-maze and the open field (thigmotactic responce) tests are two well-established behavioral procedures for the quantification of anxiety in rodents. In this study, Phobos, a novel, effective and simple application developed for recording rodents’ behavior during the elevated plus-maze and the open-field test, is being presented. Phobos is able to generate all basic locomotor-related behavioral results at once, immediately after a simple manual record of the rodent's position, along with simultaneous analysis of the experiment in 5-min periods. The efficiency of Phobos is demonstrated by presenting results from the two behavioral tests showing that animal's behavior unfolds differently in each one. Phobos manages to ease the experimenter from laborious work by providing self-explanatory characteristics and a convenient way to record the behavior of the animal, while it quickly calculates all basic locomotor-related parameters, easing behavioral studies. Phobos is freely accessible at https://sourceforge.net/projects/phobosapplication/.

Ozonation of sunflower oils: Impact of experimental conditions on the composition and the antibacterial activity of ozonized oils

Ozone can react with vegetable oils to produce ozonized oils which have antimicrobial properties and can be used in dermatology. The aim of this study was to evaluate the influence of ozonation conditions and of the initial fatty acid composition on iodine index (II), peroxide index (IP), acidity value (AV) of ozonized sunflower oils. The antibacterial activity of these products against the three bacterial strains that are more often involved in mastitis (Staphylococcus aureus, Escherichia coli and Streptococcus uberis) was also evaluated. In that purpose, two different sunflower oils have been studied: a “classical” oil (55% linoleic acid, 35% oleic acid) and a “high oleic” oil (90% oleic acid). Both were ozonized with or without water during different times (from 1 to 7h). Results show that the addition of water has a direct impact on the increase in IP (up to 2600 meq of active oxygen/kg of oil with water and 430 without) and AV but does not influence the kinetic of the decrease in II. Minimal inhibitory concentrations were ranging from 1.25 to 40mg/mL and the antibacterial activity of oils ozonized with water was better than the one of oils ozonized alone. These results are an open door to new applications of ozonized oils.

Removal of As(V) from aqueous solution by activated carbon-based hybrid adsorbents: Impact of experimental conditions

In this study, apricot stone based activated carbon (IAC) was modified with iron (oxy-hydr)oxides to produce effective hybrid adsorbents for arsenic removal from aqueous medium. For this purpose, Fe2+ loaded activated carbon (IAC-Fe(II)) and Fe3+ loaded activated carbon (IAC-Fe(III)) were produced by precipitation method. As(V) adsorption on each adsorbents was investigated at three levels of pH (3.0, 5.0 and 7.0), initial As(V) concentration (0.5, 4.5 and 8.5mgL−1) and temperature (298, 318 and 338K). As(V) adsorption capacities of IAC, IAC-Fe(II) and IAC-Fe(III) were found to be 0.034, 2.023 and 3.009mgg−1, which represented 15.00%, 98.34%, and 99.05% As(V) removal efficiency, respectively. As(V) adsorption kinetics of hybrid adsorbents were investigated and it was found that IAC-Fe(III) required less contact time than IAC-Fe(II). Thermodynamic parameters such as Gibbs free energy (ΔG°), entropy (ΔS°) and enthalpy (ΔH°) were calculated from experimental isotherm at different temperatures. The value of ΔH° for arsenate adsorption on IAC-Fe(II) was positive that indicates endothermic nature of the adsorption process. On the other hand, IAC-Fe(III) had negative value of ΔH° that indicates exothermic nature of arsenate adsorption. The equilibrium data for both hybrid adsorbents fitted well to Freundlich and Dubinin–Radushkevich. The As(V) adsorption on hybrid adsorbents had high values of correlation coefficient (R2) and low values of chi-square (χ2) for all adsorption isotherm model except Langmuir isotherm, indicate that adsorption of As(V) on hybrid adsorbents takes place on multi-layer.

Impact of Experimental Conditions on Noncontact Laser Recordings in Microvascular Studies

Microcirculation, especially skin microcirculation, is a window toward systemic vascular function in magnitude and underlying mechanisms. Different techniques have been developed to assess the microcirculation. Among these techniques, laser technology is used to perform noninvasive microvascular assessments. In the 1970s, the laser Doppler flowmetry (LDF) technique was proposed to monitor microvascular blood flow. More recently, noncontact technologies including laser Doppler perfusion imaging (LDI) and laser speckle contrast imaging (LSCI) have improved the reproducibility of the microcirculation measurements and facilitated some clinical evaluations such as on wounds and ulcers. However, due to the absence of contact between tissue and sensors, it is likely that different technical and environmental conditions may interfere with microvascular recordings. This review presents major technical and environmental conditions, which may interfere with noncontact laser recordings in microvascular studies.

X-ray structure of p38α bound to TAK-715: comparison with three classic inhibitors

The p38α mitogen-activated protein kinase regulates the synthesis of pro-inflammatory cytokines in response to stimulation by a diverse set of stress signals. Various different chemotypes and clinical candidates that inhibit p38α function have been reported over the years. In this publication, the novel structure of p38α cocrystallized with the clinical candidate TAK-715 is reported. Owing to the impact of crystallization conditions on the conformation of protein kinases (and in particular p38α), the structures of complexes of p38α with SB-203580, SCIO-469 and VX-745 have also been determined to enable in-depth comparison of ligand-induced protein conformations. The impact of experimental conditions on p38α-inhibitor complex structures, most importantly soaking versus cocrystallization, is discussed. Analysis of the structures and quantification of the protein-ligand interactions couples ligand-induced protein conformations to the number of interactions and to inhibitor selectivity against the human kinome. This shows that for the design of novel kinase inhibitors, selectivity is best obtained through maximization of the number of interactions throughout the ATP pocket and the exploitation of specific features in the active site.

Impact of experimental conditions on material response during forming of steel in semi-solid state

Semi-solid forming is an effective near-net-shape forming process to produce components with complex geometry and in fewer forming steps. It benefits from the complex thixotropic behaviour of semi-solids. However, the consequences of such behaviour on the flow during thixoforming, is still neither completely characterized and nor fully understood, especially for high melting point alloys. The study described in this paper investigates thixoextrusion for C38 low carbon steel material using dies at temperatures much lower than the slug temperature. Four different process parameters were studied: the initial slug temperature, the die temperature, the ram speed and the presence of a ceramic layer at the tool/material interface. The extruded parts were found to have an exact shape and a good surface state only if the temperature was below a certain value. This critical temperature is not an intrinsic material property since its value depends on die temperature and the presence of the Ceraspray© layer. Two kinds of flow were highlighted: a homogeneous flow controlled by the behaviour of the solid skeleton characterized by a positive strain rate sensitivity, and a non homogeneous flow (macro liquid/solid phase separation) dominated by the flow of the free liquid. With decreasing ram speed, heat losses increase so that the overall consistency of the material improves, leading to apparent negative strain rate sensitivity. Finally, some ways to optimise thixoforming are proposed.