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The nitrogen-related PTS Ntr system, present in many Proteobacteria including Escherichia coli, acts as a phosphorelay cascade composed of the EI Ntr , NPr and EIIA Ntr proteins. Phosphotransfer initiates with phosphoenolpyruvate-dependent EI Ntr autophosphorylation, the phosphoryl group is then transferred to NPr and finally to a conserved histidine residue on EIIA Ntr . The reporter metabolites l-glutamine and 2-oxoglutarate reciprocally regulate EI Ntr autophosphorylation (Lee et al., 2013) and consequently the phosphorylation status of the PTS Ntr components is controlled by the availability of nitrogen and carbon. The final phosphate acceptor, EIIA Ntr , regulates a range of cellular process by acting as the central hub of a complex protein-protein interaction network. Contact between EIIA Ntr and its target proteins is usually regulated by the EIIA Ntr phosphorylation status. In this study we performed ligand fishing assays coupled to label-free quantitative proteomics to examine the protein-protein interaction network of E. coli EIIA Ntr and a phosphomimic variant of the protein. The ligand fishing data, along with phenotypic analysis, indicated that EIIA Ntr interacts with proteins related to chemotaxis and thereby regulates cell motility. Important metabolic enzymes were also identified as potential EIIA Ntr binding partners., Competing Interests: Declaration of competing interest Authors have no conflict of interest to declare., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: Malaria is one of the most lethal and life-threatening infectious diseases in the world, causing more than half a million deaths annually. Treatment with mefloquine and artesunate is currently recommended by the World Health Organization, and was historically the first artemisinin-based combination therapy used clinically for treatment of Plasmodium falciparum. The problem of poor-quality medicines, such as counterfeit and sub-standard anti-malarials, is a worldwide issue; therefore, it is essential to develop rapid, low cost, solvent-free, and reliable methods for routine quality control for these drugs. The aim of this study was to develop and validate a novel multivariate method for direct simultaneous quantification of mefloquine and artesunate in tablets by diffuse reflectance, middle infrared spectroscopy and partial least squares regression (MIR-PLS)., Methods: Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and partial least squares regression were applied for simultaneous quantification of artesunate and mefloquine in tablets provided by the Brazilian Government. The model was obtained with full spectra (4000-400 cm(-1)) preprocessed by first derivative and Savitzky-Golay smoothing followed by mean centring, and built with three latent variables. The method was validated according to Brazilian and international guidelines through the measuring of figures of merit, such as trueness, precision, linearity, analytical sensitivity, selectivity, bias, and residual prediction deviation. The results were compared to an HPLC-MS/MS method., Results: The MIR-PLS method provided root mean square errors of prediction lower than 2.0 mg per 100 mg of powder for the two analytes, and proved to be valid according to guidelines for analytical methods that use infrared (IR) spectroscopy with multivariate calibration. For the samples obtained from Brazilian healthcare units, the method provided results statistically similar to those obtained by HPLC-MS/MS., Conclusion: MIR-PLS was found to be suitable for the quality control of these drugs. It is fast, does not use solvents, and does not generate chemical waste. Furthermore, the proposed method may be transferred and developed for use in portable equipment, increasing the possibilities for assessing the quality of these drugs.

Monitoring of the plasmatic levels of levodopa (LEV) and carbidopa (CAR) is necessary to adjust the dose of these drugs according to the individual needs of Parkinson's disease patients. To support drug therapeutic monitoring, a method using HILIC mode and LC-MS/MS was developed for the simultaneous determination of carbidopa, levodopa, and its metabolites (3-o-methyldopa (3-OMD) and dopamine (DOPA)) in human plasma. A triple quadrupole mass spectrometry was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. After straightforward sample preparation via protein precipitation, an Atlantis HILIC (150 × 2.1 mm, 3 μm, Waters, USA) column were used for separation under the isocratic condition of acetonitrile/water (79:21, v/v) containing 0.05% formic acid and 3 mmol/L ammonium formate and the total run time was 7 min. Deuterated LEV was used as internal standard for quantification. The developed method was validated in human plasma with a lower limit of quantitation of 75 ng/mL for LEV, 65 ng/mL for CAR and 3-OMD, and 20 ng/mL for DOPA. The calibration curve was linear within the concentration range of 75-800 ng/mL for LEV, 65-800 ng/mL for CAR and 3-OMD, and 20-400 ng/mL for DOPA (r>0.99). The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±13.44% of nominal and inter-assay and intra-assay precision≤13.99%. All results were within the acceptance criteria of the US FDA and ANVISA guidelines for method validation. LEV, CAR, 3-OMD and DOPA were stable in the battery of stability studies, long-term, bench-top, autosampler, and freeze/thaw cycles. Samples from patients undergoing treatment were analyzed, and the results indicated that this new method is suitable for therapeutic drug monitoring in Parkinson's disease patients., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The objective of this work was to develop and validate a HILIC-MS/MS method for the simultaneous determination of metformin and vildagliptin in human plasma. Chromatographic separation was achieved using an Atlantis HILIC Silica 150-mm × 2.1-mm, 3-μm particle size column maintained at 40°C. The isocratic mobile phase consisted of 20% water and 80% acetonitrile/water solution 95:5 (v/v), containing both 0.1% formic acid and 3mM ammonium formate. The flow rate was maintained at 400 μL min(-1). Data from validation studies demonstrated that the new method is highly selective, sensitive (limits of detection <1.5 ng mL(-1)) and free of matrix and residual effects. The new method was also precise (RSD<9.0%), accurate (RE<11.2%) and linear (r ≥ 0.99) over the ranges of 5-500 ng mL(-1) for each compound. The developed method was successfully applied to determine metformin and vildagliptin in plasma volunteers who orally received a single dose of metformin (850 mg), vildagliptin (50mg) or drug association (metformin 850 mg+vildagliptin 50mg). The new method can thus also be used as a tool for the clinical monitoring of metformin and vildagliptin., (Copyright © 2014 Elsevier B.V. All rights reserved.)