Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 g/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 g/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously ( Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a, -1b, and -2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 g/ml) lowered the ampicillin MIC to 3.67 g/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.

Nociceptive neurons play an essential role in pain sensation by transmitting painful stimuli to the central nervous system. However, investigations of nociceptive neuron biology have been hampered by the lack of accessibility of human nociceptive neurons. Here, we describe a system for efficiently guiding human embryonic stem cells into nociceptive neurons by first inducing these cells to the neural lineage. Subsequent addition of retinoic acid and BMP4 at specific time points and concentrations yielded a high population of neural crest progenitor cells (AP2α+, P75+), which further differentiated into nociceptive neurons (TRKA+, Nav1.7+, P2X3+). The overexpression of Neurogenin 1 (Neurog1) promoted the neurons to express genes related to sensory neurons (Peripherin, TrkA) and to further mature into TRPV1+ nociceptive neurons. Importantly, the overexpression of Neurog1 increased the response of these neurons to capsaicin stimulation, a hallmark of mature functional nociceptive neurons. Taken together, this study reveals the important role that Neurog1 plays in generating functional human nociceptive neurons.

High-throughput screening for drug discovery is increasingly utilizing cellular systems of high physiological relevance, such as patient primary cells and organoid cultures. We used 3D-cultured cystic fibrosis patient bronchial epithelial cells to screen for new small-molecule correctors of the disease-causing F508del mutation in CFTR. Impaired mucociliary clearance due to insufficient airway hydration is a hallmark of cystic fibrosis and we used a simple measure of surface liquid levels to quantify F508del CFTR correction in cultured bronchial epithelial cells. Two robust assay formats were configured and used to screen more than 100,000 compounds as mixtures or individual compounds in 96-well format. The corrector discovery success rate, as measured by the number of hits confirmed by an electrophysiology assay on patient primary bronchial epithelial cells, was superior to screens in cell lines expressing recombinant F508del CFTR. Several novel corrector scaffolds were discovered that when combined with the clinical corrector VX-809 delivered combination responses greater than double that of VX-809 alone. This work exemplifies the advantages of a disease-relevant readout and 3D-cultured patient primary cells for the discovery of new drug candidates.

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