Drug efflux and lipid A modification by 4-L-aminoarabinose are key mechanisms of polymyxin B resistance in the sepsis pathogen Enterobacter bugandensis.

OBJECTIVES: A concern with the ESKAPE pathogen, Enterobacter bugandensis, and other species of the Enterobacter cloacae complex, is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins. METHODS: Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis. RESULTS: In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain, W3110. The efflux pump, AcrABTolC, and the two component regulators, PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modelling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps. CONCLUSION: Our results support a model where drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.

Enfortumab vedotin and pembrolizumab as monotherapies and combination treatment in locally advanced or metastatic urothelial carcinoma: A narrative review.

Background: Bladder cancer is the 10th most common cancer globally. The majority of bladder cancers are urothelial carcinomas (UCs), which, if locally advanced or metastatic, carry poor long-term prognosis. Cancer cells can evade the immune system by expressing the programmed cell death ligand 1 protein (PD-L1). Programmed cell death ligand 1 protein binds to programmed cell death protein 1 (PD-1) on T cells, inhibiting their antitumor action. Bladder tumor cells also overexpress nectin-4, a cell adhesion polypeptide that contributes to metastasis, worsening prognosis. Current platinum-based chemotherapy treatments are suboptimal. This review aimed to assess novel treatments for locally advanced or metastatic UC that specifically target PD-L1 or nectin-4, namely, the PD-1 inhibitor pembrolizumab and the anti-nectin-4 antibody-drug conjugate enfortumab vedotin (EV). Materials and methods: Relevant English-language peer-reviewed articles and conference abstracts from the last 5 years were identified through MEDLINE and EMBASE database searches. A narrative review was performed, with key results outlined below. Results: Pembrolizumab was demonstrated to be superior to chemotherapy as a second-line treatment for platinum-unresponsive participants in the KEYNOTE-045 trial, resulting in its Food and Drug Administration (FDA) approval. Enfortumab vedotin therapy resulted in superior outcomes compared with chemotherapy in the EV-301 trial, resulting in FDA approval for its use for patients with locally advanced or metastatic UC who had previously undergone treatment with platinum-based chemotherapy and PD-1/PD-L1 inhibitors. Positive preliminary results for pembrolizumab and EV combination therapy have led to FDA approval in patients with locally advanced or metastatic UC who are not eligible for platinum chemotherapy. Conclusions: Pembrolizumab and EV represent novel treatment options for patients with locally advanced or metastatic UC with documented superior outcomes and tolerability as compared with standard chemotherapy.

Utilisation and valorisation of distillery whisky waste streams via biomass electrolysis: electrosynthesis of hydrogen.

Fuel-flexible hydrogen generation methods, such as electrochemical conversion of biomass, offer a route for sustainable production of hydrogen whilst valorising feedstocks that are often overlooked as waste products. This work explores the potential of a novel, two-stage electrolysis process to convert biomass-containing solid (draff/spent barley) and liquid (pot ale and spent lees) whisky co-products, from the Isle of Raasay Distillery, into hydrogen, using a phosphomolybdic acid (H3[PMo12O40] or PMA) catalyst. Characterisation results for whisky distillery co-products will be presented, including thermogravimetric, differential scanning calorimetric, CHN elemental, total organic carbon and chemical oxygen demand analysis data. The results indicated that the characteristics of these co-products align well with those reported across the Scotch whisky distillation sector. Subsequently, the concept of thermal digestion of each co-product type, using the Keggin-type polyoxometalate PMA catalyst to abstract protons and electrons from biomass, will be outlined. UV-visible spectrophotometry was employed to assess the extent of reduction of the catalyst, after digestion of each co-product, and indicated that draff and pot ale offer the largest scope for hydrogen production, whilst digestion and electrolysis of spent lees is not viable due to the low biomass content of this distillation co-product. Finally, details of electrolysis of the PMA-biomass solutions using a proton-exchange membrane electrolysis cell (PEMEC) will be provided, including electrochemical data that help to elucidate the performance-limiting processes of the PEMEC operating on digested biomass-PMA anolytes.

Polymyxin Resistance and Heteroresistance Are Common in Clinical Isolates of Achromobacter Species and Correlate with Modifications of the Lipid A Moiety of Lipopolysaccharide.

The Achromobacter genus includes opportunistic pathogens that can cause chronic infections in immunocompromised patients, especially in people with cystic fibrosis (CF). Treatment of Achromobacter infections is complicated by antimicrobial resistance. In this study, a collection of Achromobacter clinical isolates, from CF and non-CF sources, was investigated for polymyxin B (PmB) resistance. Additionally, the effect of PmB challenge in a subset of isolates was examined and the presence of PmB-resistant subpopulations within the isolates was described. Further, chemical and mass spectrometry analyses of the lipid A of Achromobacter clinical isolates enabled the determination of the most common structures and showed that PmB challenge was associated with lipid A modifications that included the addition of glucosamine and palmitoylation and the concomitant loss of the free phosphate at the C-1 position. This study demonstrates that lipid A modifications associated with PmB resistance are prevalent in Achromobacter and that subresistant populations displaying the addition of positively charged residues and additional acyl chains to lipid A can be selected for and isolated from PmB-sensitive Achromobacter clinical isolates. IMPORTANCE Achromobacter species can cause chronic and potentially severe infections in immunocompromised patients, especially in those with cystic fibrosis. Bacteria cannot be eradicated due to Achromobacter's intrinsic multidrug resistance. We report that intrinsic resistance to polymyxin B (PmB), a last-resort antimicrobial peptide used to treat infections by multiresistant bacteria, is prevalent in Achromobacter clinical isolates; many isolates also display increased resistance upon PmB challenge. Analysis of the lipopolysaccharide lipid A moiety of several Achromobacter species reveals a penta-acylated lipid A, which in the PmB-resistant isolates was modified by the incorporation of glucosamine residues, an additional acyl chain, loss of phosphates, and hydroxylation of acyl chains, all of which can enhance PmB resistance in other bacteria. We conclude that PmB resistance, particularly in Achromobacter isolates from chronic respiratory infections, is a common phenomenon, and that Achromobacter lipid A displays modifications that may confer increased resistance to polymyxins and potentially other antimicrobial peptides.

Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons.

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

The role of protein complexes in human genetic disease.

Many human genetic disorders are caused by mutations in protein-coding regions of DNA. Taking protein structure into account has therefore provided key insight into the molecular mechanisms underlying human genetic disease. Although most studies have focused on the intramolecular effects of mutations, the critical role of the assembly of proteins into complexes is being increasingly recognized. Here, we review multiple ways in which consideration of protein complexes can help us to understand and explain the effects of pathogenic mutations. First, we discuss disorders caused by mutations that perturb intersubunit interactions in homomeric and heteromeric complexes. Second, we address how protein complex assembly can facilitate a dominant-negative mechanism, whereby mutated subunits can disrupt the activity of wild-type protein. Third, we show how mutations that change protein expression levels can lead to damaging stoichiometric imbalances. Finally, we review how mutations affecting different subunits of the same heteromeric complex often cause similar diseases, whereas mutations in different interfaces of the same subunit can cause distinct phenotypes.

Selective hydrogenation of nitroarenes using an electrogenerated polyoxometalate redox mediator.

The 2-electron reduced form of the polyoxometalate silicotungstic acid (H4[SiW12O4]) is shown to be an effective and selective hydrogenation agent for a range of nitroarenes without the need for any co-catalyst. The ease of generation of the active species and its recyclability suggest that a new approach to this important class of chemical conversions is possible.