Purpose: Several mass spectrometry-based methods for antimicrobial sensitivity testing have been described in recent years. They offer an alternative to commercially available testing systems which were considered to have disadvantages in terms of cost- and time-efficiency. The aim of this study was to develop an LC-MS/MS-based antibiotic hydrolysis assay for evaluating antimicrobial resistance (AMR) of Gram-negative bacteria. Materials and Methods: Four species of Gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Providencia stuartii and Acinetobacter baumannii) were tested against six antibiotics from three different classes: ampicillin, meropenem, imipenem, ceftazidime, ceftriaxone and cefepime. Bacterial suspensions from each species were incubated with a mixture of the six antibiotics. Any remaining antibiotic following incubation was measured using LC-MS/MS. The results were interpreted using measurements obtained for an E. coli strain sensitive to all antibiotics and expressed as percentage of hydrolyzed antibiotic. These were subsequently compared to commercially-available system for the bacteria identification and susceptibility testing. Results: Overall, LC-MS/MS assay and commercial antimicrobial susceptibility platform results showed good agreement in terms of an organism being resistant/sensitive to an antibiotic. The time required to complete the LC-MS/MS-based hydrolysis test was under 5 h, significantly shorter that commercially available susceptibility testing platforms. Conclusion: By using a sensitive strain for results interpretation and simultaneous use of multiple antibiotics, the proposed protocol offers improved robustness and multiplexing over previously described methods for antibiotic sensitivity testing. Nevertheless, further research is needed before routine assimilation of the method, especially for strains with intermediate resistance.

Resistance to imatinib, a first-line BCR-ABL1 tyrosine kinase inhibitor for chronic myeloid leukemia, is frequently mediated by drug efflux through P-glycoprotein (P-gp) overexpression. We report the design, synthesis, and evaluation of eight novel imatinib derivatives modified at the piperazine terminus with efflux resistance breaker (ERB) fragments to reduce P-gp-mediated efflux. In silico docking against cryo-EM P-gp structures predicted increased hydrophobic interactions and enhanced occupancy at the access tunnel, indicative of efflux inhibition. Compound 8 showed potency comparable to imatinib in BCR-ABL1+ K562 cells and a lower LC50 fold change in resistant K562/DOX cells, suggesting reduced efflux susceptibility. Accumulation assays confirmed the improved intracellular retention of compound 8. Compound 9 displayed increased potency in resistant cells, correlating with higher intracellular levels despite modest kinase inhibition. Verapamil assays confirmed reduced efflux liability for compounds 8 and 13. Compound 8 also showed a positive therapeutic index. These findings support rational design to mitigate efflux-mediated resistance.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.