A hybrid strategy combining solution NMR spectroscopy and isothermal titration calorimetry to characterize protein-nanodisc interaction.

NMR is a powerful tool for characterizing intermolecular interactions at atomic resolution. However, the nature of the complex interactions of membrane-binding proteins makes it difficult to elucidate the interaction mechanisms. Here, we demonstrated that structural and thermodynamic analyses using solution NMR spectroscopy and isothermal titration calorimetry (ITC) can clearly detect a specific interaction between the pleckstrin homology (PH) domain of ceramide transport protein (CERT) and phosphatidylinositol 4-monophosphate (PI4P) embedded in the lipid nanodisc, and distinguish the specific interaction from nonspecific interactions with the bulk surface of the lipid nanodisc. This NMR-ITC hybrid strategy provides detailed characterization of protein-lipid membrane interactions.

An environment-friendly spot test method with digital imaging for the micro-titration of citric fruits.

A new method to determine the total titratable acidity of orange, lemon and passion fruit, based on a spot test obtained from digital images and using anthocyanins as the biodegradable indicator, is presented for the first time. The colorimetric reactions were carried out by acid-base titration on a microscale, employing anthocyanin with a microplate for spot test purposes, with detection by digital imaging. To obtain highly precise data, a chamber based on a diffuser was developed to control the illumination supplied by the light emitting diodes, and coupled to a smartphone to acquire adequate digital images. High precision was obtained with a relative standard deviation of 0.758% for n = 95. The RGB values were extracted from the digital images and used as analytical signals, the values being correlated with the micro-volume of the titrant and used to construct the titration curves and obtain the first and second derivatives, respectively. For comparative purposes, the official AOAC (Association of Official Analytical Chemists) and MAPA (Ministry of Agriculture, Livestock and Food Supply of Brazil) methods were used and the results compared by applying the paired t-test at the 95% confidence level (n = 3). No difference was found between the values and the relative errors were less than 2.8%. The micro-titrimetric method was fast, uses anthocyanins as the natural indicator, is practical, and permits a reduction of 922 times or 99.9% of the volume required in a conventional titration. It is therefore ideal for routine analyses leading to a reduction in the waste generated, according to the principles of green chemistry.

Barriers and Facilitators to the Implementation of a Mobile Insulin Titration Intervention for Patients With Uncontrolled Diabetes: A Qualitative Analysis.

BACKGROUND: In 2016, a short message service text messaging intervention to titrate insulin in patients with uncontrolled type 2 diabetes was implemented at two health care facilities in New York City. OBJECTIVE: This study aimed to conduct a qualitative evaluation assessing barriers to and the facilitators of the implementation of the Mobile Insulin Titration Intervention (MITI) program into usual care. METHODS: We conducted in-depth interviews with 36 patients enrolled in the MITI program and the staff involved in MITI (n=19) in the two health care systems. Interviews were transcribed and iteratively coded by two study investigators, both inductively and deductively using a codebook guided by the Consolidated Framework for Implementation Research. RESULTS: Multiple facilitator themes emerged: (1) MITI had strong relative advantages to in-person titration, including its convenience and time-saving design, (2) the free cost of MITI was important to the patients, (3) MITI was easy to use and the patients were confident in their ability to use it, (4) MITI was compatible with the patients' home routines and clinic workflow, (5) the patients and staff perceived MITI to have value beyond insulin titration by reminding and motivating the patients to engage in healthy behaviors and providing a source of patient support, and (6) implementation in clinics was made easy by having a strong implementation climate, communication networks to spread information about MITI, and a strong program champion. The barriers identified included the following: (1) language limitations, (2) initial nurse concerns about the scope of practice changes required to deliver MITI, (3) initial provider knowledge gaps about the program, and (4) provider perceptions that MITI might not be appropriate for some patients (eg, older or not tech-savvy). There was also a theme that emerged during the patient and staff interviews of an unmet need for long-term additional diabetes management support among this population, specifically diet, nutrition, and exercise support. CONCLUSIONS: The patients and staff were overwhelmingly supportive of MITI and believed that it had many benefits and that it was compatible with the clinic workflow and patients' lives. Initial implementation efforts should address staff training and nurse concerns. Future research should explore options for integrating additional diabetes support for patients.

Electrophoresis Titration Model of a Moving Redox Boundary Chip for a Point-of-Care Test of an Enzyme-Linked Immunosorbent Assay.

Enzyme-linked immunosorbent assays (ELISAs) have been widely used in clinical examination, food safety, and environmental analyses. However, they still face a great challenge in designing a device for a point-of-care test (POCT) due to its bulk optical detector and complexity. Herein an electrophoresis titration (ET) model of a moving redox boundary (MRB) was proposed for constructing an ET-ELISA chip of a POCT just with sextuplet electrode pairs and laminated cells. The chip had an anodic well, middle well, and cathode well which were connected by microchannels. The ELISA process was conducted in the bottom of the middle well, where horseradish peroxidase (HRP) catalyzed 3,3',5,5'-tetra-methyl benzidine (TMB) as a blue TMB dimer with two positive charges. Under an electrical field of 29 V, the TMB dimer migrated into the titration channel and reacted with the ascorbic acid, creating an MRB. The MRB motion was a function of antigen content, indicating a visual distance-based assay. As a proof of concept, a C-reactive protein was chosen as a model antigen. The experiments systemically validated the ET-ELISA model and method. Particularly, the chip was smartphone-detected, traditional power supply free, and did not use sulfuric acid used in typical ELISA, making the ET-ELISA method extremely simple, portable, and safe. The ET-ELISA has great potential to visual and portable ELISA in clinical medicine, the environment, and food safety immunoassay.

Filling Blank Spots on the Map: Identification of Ligand Binding Modes and Interacting Water Molecules for Brd4-BD1 by WaterLOGSY Titrations.

Fragment-based drug discovery and continuous improvement of existing protein inhibitors rely on the knowledge of exactly how and how strongly a range of small molecules bind to their respective protein targets. By increasing the (perdeuterated) protein concentration, WaterLOGSY titration experiments give access to ligand binding modes even in the case of weak binders as well as to the location of protein-bound water in the surroundings of the ligand. On the basis of these findings, specific chemical modifications of the ligand could be shown to yield significantly enhanced binding affinities.

Massively parallel and multiparameter titration of biochemical assays with droplet microfluidics.

Biochemical systems in which multiple components take part in a given reaction are of increasing interest. Because the interactions between these different components are complex and difficult to predict from basic reaction kinetics, it is important to test for the effect of variations in the concentration for each reagent in a combinatorial manner. For example, in PCR, an increase in the concentration of primers initially increases template amplification, but large amounts of primers result in primer-dimer by-products that inhibit the amplification of the template. Manual titration of biochemical mixtures rapidly becomes costly and laborious, forcing scientists to settle for suboptimal concentrations. Here we present a droplet-based microfluidics platform for mapping of the concentration space of up to three reaction components followed by detection with a fluorescent readout. The concentration of each reaction component is read through its internal standard (barcode), which is fluorescent but chemically orthogonal. We describe in detail the workflow, which comprises the following: (i) production of the microfluidics chips, (ii) preparation of the biochemical mixes, (iii) their mixing and compartmentalization into water-in-oil emulsion droplets via microfluidics, (iv) incubation and imaging of the fluorescent barcode and reporter signals by fluorescence microscopy and (v) image processing and data analysis. We also provide recommendations for choosing the appropriate fluorescent markers, programming the pressure profiles and analyzing the generated data. Overall, this platform allows a researcher with a few weeks of training to acquire ∼10,000 data points (in a 1D, 2D or 3D concentration space) over the course of a day from as little as 100-1,000 µl of reaction mix.

Development of a Platform to Enable Fully Automated Cross-Titration Experiments.

In the triage of hits from a high-throughput screening campaign or during the optimization of a lead compound, it is relatively routine to test compounds at multiple concentrations to determine potency and maximal effect. Additional follow-up experiments, such as agonist shift, can be quite valuable in ascertaining compound mechanism of action (MOA). However, these experiments require cross-titration of a test compound with the activating ligand of the receptor requiring 100-200 data points, severely limiting the number tested in MOA assays in a screening triage. We describe a process to enhance the throughput of such cross-titration experiments through the integration of Hewlett Packard's D300 digital dispenser onto one of our robotics platforms to enable on-the-fly cross-titration of compounds in a 1536-well plate format. The process handles all the compound management and data tracking, as well as the biological assay. The process relies heavily on in-house-built software and hardware, and uses our proprietary control software for the platform. Using this system, we were able to automate the cross-titration of compounds for both positive and negative allosteric modulators of two different G protein-coupled receptors (GPCRs) using two distinct assay detection formats, IP1 and Ca2+ detection, on nearly 100 compounds for each target.

Lab on paper: iodometric titration on a printed card.

A paper test card has been engineered to perform an iodometric titration, an application that requires storage and mixing on demand of several mutually incompatible reagents. The titration is activated when a user applies a test solution to the test card: the dried reagents are reconstituted and combined through a surface-tension-enabled mixing (STEM) mechanism. The device quantifies 0.8-15 ppm of iodine atoms from iodate in aqueous solutions. This is useful, for example, to quantify iodine levels in fortified salt. A blinded internal laboratory validation established the accuracy as 1.4 ppm I and the precision as 0.9 ppm I when the test card was read by newly trained users. Using computer software to process images, the accuracy and precision both improved to 0.9 ppm I. The paper card can also detect substandard ß lactam antibiotics using an iodometric back-titration. When used to quantify amoxicillin, good distinction is achieved between solutions that differ by 0.15 mg/mL over a working range of 0-0.9 mg/mL. The test card was designed to meet the World Health Organization ASSURED criteria for use in low resource settings, where laboratory-based analytical procedures are often not available.

Coulometric titration with electrogenerated oxidants as a tool for evaluation of cognac and brandy antioxidant properties.

Stoichiometric coefficients for reactions of cognac antioxidants with coulometric titrants (electrogenerated bromine and hexacyanoferrate(III) ions) have been found. Ellagic and gallic acids react with both titrants while aldehydes (vanillin, syringic and coniferaldehyde) - with electrogenerated bromine only. Furfurals do not show significant reactivity toward both oxidants. Cognac and brandy total antioxidant capacity (TAC) and ferric reducing power (FRP) based on reactions with electrogenerated bromine and hexacyanoferrate(III) ions, respectively, have been evaluated. Both parameters for cognacs are statistically significant higher than for brandies and grow with the age increase. Beverages under investigation has shown relatively high antiradical activity toward 2,2-diphenyl-1-picrylhydrazyl (7-92% and 5-93% for cognacs and brandies, respectively). Total phenolics content has been evaluated by Folin-Ciocalteu method. Older beverages represent the higher phenolics content caused by more time of extraction from oak barrels. Positive correlations (r=0.8077-0.9617) have been observed for TAC and FRP with antiradical activity and total phenolics content.

Automated spectrophotometric analyzer for rapid single-point titration of seawater total alkalinity.

An automated analyzer was developed to achieve fast, precise, and accurate measurements of seawater total alkalinity (AT) based on single-point titration and spectrophotometric pH detection. The single-point titration was carried out in a circulating loop, which allowed the titrant (hydrochloric acid and bromocresol green solution) and a seawater sample to mix at a constant volume ratio. The dissolved CO2 in the sample-titrant mixture was efficiently removed by an inline CO2 remover, which consists of a gas-permeable tubing (Teflon AF2400) submerged in a sodium hydroxide (NaOH) solution. The pH of the mixture was then measured with a custom-made spectrophotometric detection system. The analyzer was calibrated against multiple certified reference materials (CRMs) with different AT values. The analyzer features a sample throughput time of 6.5 min with high precision (±0.33-0.36 µmol kg(-1); n = 48) and accuracy (-0.33 ± 0.99 µmol kg(-1); n = 10). Intercomparison to a traditional open-cell AT titrator showed overall good agreement of 0.88 ± 2.03 µmol kg(-1) (n = 22). The analyzer achieved excellent stability without recalibration over 11 days, during which time 320 measurements were made with a total running time of over 40 h. Because of its small size, low power consumption requirements, and its ability to be automated, the new analyzer can be adapted for underway and in situ measurements.

A visual detection of protein content based on titration of moving reaction boundary electrophoresis.

A visual electrophoretic titration method was firstly developed from the concept of moving reaction boundary (MRB) for protein content analysis. In the developed method, when the voltage was applied, the hydroxide ions in the cathodic vessel moved towards the anode, and neutralized the carboxyl groups of protein immobilized via highly cross-linked polyacrylamide gel (PAG), generating a MRB between the alkali and the immobilized protein. The boundary moving velocity (V(MRB)) was as a function of protein content, and an acid-base indicator was used to denote the boundary displacement. As a proof of concept, standard model proteins and biological samples were chosen for the experiments to study the feasibility of the developed method. The experiments revealed that good linear calibration functions between V(MRB) and protein content (correlation coefficients R>0.98). The experiments further demonstrated the following merits of developed method: (1) weak influence of non-protein nitrogen additives (e.g., melamine) adulterated in protein samples, (2) good agreement with the classic Kjeldahl method (R=0.9945), (3) fast measuring speed in total protein analysis of large samples from the same source, and (4) low limit of detection (0.02-0.15 mg mL(-1) for protein content), good precision (R.S.D. of intra-day less than 1.7% and inter-day less than 2.7%), and high recoveries (105-107%).

Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis.

In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross-linked polyacrylamide gel (PAG), an acid-base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method.

Considerations in determination of residual moisture in lyophilized demineralized bone matrix: the role of residual moisture analyzers.

The objective of this study is to determine whether a residual moisture analyzer (RMA) can be an acceptable instrument for measuring the residual moisture in lyophilized demineralized bone matrix (DBM). Instruments from two different manufacturers with differing configurations and controls were compared: the Ohaus MB45 and Arizona Instrument MAX4000XL. The effects of various factors such as test temperature, drying profile, end point criteria, lift compensation, chamber configuration, and rehydration on residual moisture (RM) are examined. The performance of the RMAs is based on their ability to reproduce RM results obtained by the current standard gravimetric method. RMAs provide reliable, accurate and reproducible results in a number of industries that rely on the determination of RM. We hypothesize that RMAs are suitable for measuring RM in DBM and provide validation study data with optimized settings for these two instruments. Potentially, such studies will provide justification for allowance of this methodology as an acceptable alternative to the current gravimetric method allowed by American Association of Tissue Banks Standards.

Isothermal titration calorimetry as a tool to determine the thermodynamics of demicellization processes.

Demicellization of a 90 mM sodium dodecyl sulfate (SDS) solution in water at 10, 22, and 30 °C was studied by isothermal titration calorimetry (ITC). ΔH of the demicellization process was strongly temperature dependent, having an exothermic progression (-20.4 ± 0.9 kJ∕mol, max) at 10 °C and an endothermic one (3.7 ± 1.2 kJ∕mol, max) at 30 °C. ΔH for micelle dilution followed a slightly endothermic progression (0.9 ± 0.5 kJ∕mol at 30 °C, 0.7 ± 1.3 kJ∕mol at 22 °C, and 0.0 ± 0.5 kJ∕mol at 10 °C) at all studied temperatures. No differences in ΔH for micelle dilution and demicellization was observed at 22 °C. The temperature dependence of ΔH measured by ITC could be related to hydrophobic interactions. Therefore, ITC was shown to be a useful tool to describe the thermodynamics of demicellization processes and in addition to determine alterations in ΔH caused by changes in hydrophobic and steric∕electrostatic interactions.

Electroanalytical measurements without electrolytes: conducting polymers as probes for redox titration in non-conductive organic media.

Electroanalytical methods have been applied only in conducting media. An application of conducting polymers allows to overcome this limitation. If such material is in electrochemical equilibrium with dissolved redox active species, its electrical conductivity depends on the redox potential of these species. Therefore, conductometric measurements with conducting polymers can provide about the same information as classical redox electrodes. The approach was applied for redox titration. Equivalent points obtained by this titration in aqueous and organic electrolytes were identical. Then the approach was applied for determination of bromine number by redox titration in non-conducting organic phase.

Nonlinear analyte concentration gradients for one-step kinetic analysis employing optical microring resonators.

Conventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes, and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics.

[Adaptation of methotrexate determination in serum with Unicel DxC600(®)]. / Adaptation du dosage sérique du méthotrexate sur Unicel DxC600(®).

High-dose methotrexate treatment requires pharmacological monitoring in order to tailor administration of folinic acid to reduce side effects. The aim of the study was to validate the adaptation of the EMIT reagent on the l'Unicel DxC 600® Beckman Coulter. The establishment of two assays was necessary to obtain a quantification limit as low as possible (0.05 µmol/L). The linearity of the adapted methods extends from 0.05 to 0.25 µmol/L on the one hand, and from 0.25 to 1 µmol/L on the other hand. For each method, fidelity and accuracy were studied and the limits of detection and quantification were quantified. The correlation with the FPIA method was performed on the Abbott TDX(®). The results of all tests are satisfactory with coefficients of variation (CV) of repeatability and reproducibility of less than 6%. However the daily assays are heavy as 66% of blood samples require at least two dosages and 30% a manual dilution.

Flexible diuretic titration in chronic heart failure: where is the evidence?

BACKGROUND: Several sets of heart failure (HF) consensus/guideline statements support the use of a flexible diuretic dosing regimen for HF outpatient management of fluid overload-related signs and symptoms. However, despite the widespread acceptance of such an approach, the evidence supporting the effectiveness of this approach in improving clinical outcomes is unknown. The primary objective of this manuscript was to summarize and review the evidence supporting the use of a flexible diuretic regimen in the management of outpatient heart failure patients. METHODS AND RESULTS: A systematic review was performed, and 9 studies were identified relevant to the question of flexible diuretic titration in the setting of chronic heart failure. Among the 9 studies, 5 were randomized. Three of the randomized trials included flexible diuretic titration as part of a broader multifaceted disease management program, and only 2 were designed to specifically evaluate the sole contribution of flexible diuretic titration. Collectively, data from all of the studies reviewed supported the idea that flexible and individualized diuretic dosing is potentially associated with reduced emergency room visits, reduced rehospitalization, and improved quality of life in HF patients with reduced ejection fraction. CONCLUSIONS: To date, only 2 randomized clinical studies were identified that were designed to determine the effects of a flexible diuretic dosing regimen in outpatient HF patients with reduced ejection fraction. Data are lacking in HF patients with preserved ejection fraction. There is a critical need to test this strategy in well designed prospective randomized clinical trials.

Potentiometric determination of cetylpyridinium chloride using a new type of screen-printed ion selective electrodes.

A new type of screen-printed ion-selective electrode for the determination of cetylpyridinium chloride (CPC) is presented. These new electrodes involve in situ, modified and unmodified screen-printed ion-selective electrodes for the determination of CPC. The screen-printed electrodes (SPEs) show a stable, near-Nernstian response for 1 x 10(-2) to 1 x 10(-6) M CPC at 25 degrees C over the pH range 2-8 with cationic slope 60.66+/-1.10. The lower detection limit is found to be 8 x 10(-7) M and response time of about 3s and exhibit adequate shelf-life (6 months). The fabricated electrodes can be also successfully used in the potentiometric titration of CPC with sodium tetraphenylborate (NaTPB). The analytical performances of the SPEs are compared with those for carbon paste electrode (CPE) and polyvinyl chloride (PVC) electrodes. The method is applied for pharmaceutical preparations with a percentage recovery of 99.60% and R.S.D.=0.53. The frequently used CPC of analytical and technical grade as well as different water samples has been successfully titrated and the results obtained agreed with those obtained with commercial electrode and standard two-phase titration method. The sensitivity of the proposed method is comparable with the official method and ability of field measurements.

The 'strip method': a simple method for plaque pH assessment.

AIM: The aim of the study was to evaluate if pH indicator strips could be used for measurements of plaque pH acidogenicity in situ. METHOD: Interproximal plaque pH was measured before and up to 60 min after a 10% sucrose rinse in 30 healthy volunteers using pH indicator strips and the microtouch method in parallel. RESULTS: It was found that the 'strip method' could determine changes in plaque pH to the same extent as the microtouch method (correlation coefficient 0.99). CONCLUSION: Since the 'strip method' is inexpensive and easy to handle, it may be applicable for assessment of plaque acidogenicity in the clinic.