The phage-derived phiC31 integrase is a useful tool for mediating sequence-specific genomic integration in mammalian cells, recombining donor plasmids bearing the attB recognition site with introduced genomic attP sites or endogeneous pseudo-attP sites having partial identity to attP. In most prior studies, phiC31 integrase has been introduced as plasmid DNA or mRNA. The current report examines whether phiC31 integrase functions efficiently in mammalian cells when co-nucleofected as a purified protein, along with attB-containing donor plasmids or PCR fragments. We describe preparation of phiC31 integrase protein and evidence that it can mediate genomic integration in human 293 cells, including PCR evidence for integration at an endogenous pseudo-attP site. This work demonstrates for the first time the ability of 605- and 613-amino-acid versions of phiC31 integrase protein to mediate efficient, site-specific integration into the genome of human cells when co-nucleofected with full-sizedattB-containing donor plasmids or linear 2.5-kb PCR fragments. VAV1 degrader-3 solubility dmso This protein-mediated approach may be especially useful for integration of exogenous sequences into valuable therapeutic target cells, such as hematopoietic stem cells or T cells, that are sensitive to introduced DNA. Pseudomonas aeruginosa is a priority pathogen for the development of new antibiotics, particularly because multi-drug resistant strains of this bacterium cause serious nosocomial infections and are the leading cause of death in cystic fibrosis patients. Pyocins, bacteriocins of P. aeruginosa, are potent and diverse protein antibiotics that are deployed during bacterial competition. Pyocins are produced by more than 90% of P. aeruginosa strains and may have utility as last resort antibiotics against this bacterium. In this study, we explore the antimicrobial activity of a newly discovered pyocin called pyocin G (PyoG). We demonstrate that PyoG has broad killing activity against a collection of clinical P. aeruginosa isolates and is active in a Galleria mellonella infection model. We go on to identify cell envelope proteins that are necessary for the import of PyoG and its killing activity. PyoG recognises bacterial cells by binding to Hur, an outer membrane TonB dependent transporter. Both pyocin and Hur interact with TonB1, which in complex with ExbB-ExbD links the proton motive force generated across the inner membrane with energy dependent pyocin translocation across the outer membrane. Inner membrane translocation of PyoG is dependent on the conserved inner membrane AAA+ ATPase/protease, FtsH. We also report a functional exploration of the PyoG receptor. We demonstrate that Hur can bind to hemin in vitro and that this interaction is blocked by PyoG, confirming the role of Hur in hemin acquisition. Modulating the abundance of the blood-brain barrier (BBB) efflux transporter breast cancer resistance protein (BCRP) has the potential to impact brain levels of drugs and endogenous substrates. Studies have demonstrated that the metal ionophore clioquinol (CQ) increases BBB abundance of P-glycoprotein (P-gp), an effect associated with increased endothelial cell levels of Cu2+. This study therefore assessed whether human brain endothelial (hCMEC/D3) cell abundance and function of BCRP is modulated by CQ. hCMEC/D3 cells were treated with CQ, Zn2+ and Cu2+ (CZC) (0.5 μM, 0.5 μM, 0.1 μM, respectively) for 24 h and BCRP mRNA and protein abundance was determined by Western blot and qPCR, respectively. After a series of optimisation studies assessing specificity of bodipy prazosin (BP) and Ko143 as a substrate and inhibitor of BCRP, respectively, the impact of CZC on BP uptake was assessed. While CZC did not increase mRNA expression of BCRP, BCRP abundance was increased 1.8±0.1-fold; this was associated with a 68.1±3.3% reduction in accumulation of BP in hCMEC/D3 cells. This is the first study to demonstrate that augmenting metal ion availability enhances protein abundance and function of BCRP at the BBB, which may be exploited to modulate CNS access of therapeutics and endogenous substrates. Equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective nucleobase transporter. Although it is highly expressed in the liver, its role in nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 μM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 μM, which was comparable to that of 2.30 μM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells. Drug products containing the antibiotics amoxicillin (500 mg as trihydrate) or doxycycline (200 mg as hyclate or monohydrate) with varying qualitative excipient composition were obtained from the German market and their biopharmaceutical properties were characterized in compendial quality control tests, dissolution tests run under BCS-based biowaiver conditions and dissolution tests using biorelevant media. Observed differences in disintegration time and dissolution rate were assessed according to BCS-based biowaiver dissolution specifications and in virtual bioequivalence trials using GastroPlusTM. Great variation was observed in dosage form performance, and two out of five drug products for each active ingredient failed to demonstrate in vitro similarity using the BCS-based biowaiver specifications, with coning being identified as a key hindrance. Nonetheless, all drug products investigated were found to be equivalent in virtual trials, concordant with their market approval status, indicating that the current BCS-based biowaiver criteria are over-discriminating.