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Disclosure and competing interests statement: Brunel University London has two patents covering the therapeutic use of artificial sweeteners and their use to potentiate antibiotic activity. Data availability The RNA-seq datasets produced in this study (gene expression dataset series titled "Alteration of global transcription by the artifi- cial sweetener acesulfame-K in Acinetobacter baumannii AB5075") are available at the National Center for Biotechnology Information Gene Expression Omnibus public database under accession number GSE199706 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE199706). Copyright © 2022 The Authors. Antimicrobial resistance is one of the most pressing concerns of our time. The human diet is rich with compounds that alter bacterial gut communities and virulence-associated behaviours, suggesting food additives may be a niche for the discovery of novel anti-virulence compounds. Here, we identify three artificial sweeteners, saccharin, cyclamate and acesulfame-K (ace-K), that have a major growth inhibitory effect on priority pathogens. We further characterise the impact of ace-K on multidrug-resistant Acinetobacter baumannii, demonstrating that it can disable virulence behaviours such as biofilm formation, motility and the ability to acquire exogenous antibiotic-resistant genes. Further analysis revealed the mechanism of growth inhibition is through bulge-mediated cell lysis and that cells can be rescued by cation supplementation. Antibiotic sensitivity assays demonstrated that at sub-lethal concentrations, ace-K can resensitise A. baumannii to last resort antibiotics, including carbapenems. Using a novel ex vivo porcine skin wound model, we show that ace-K antimicrobial activity is maintained in the wound microenvironment. Our findings demonstrate the influence of artificial sweeteners on pathogen behaviour and uncover their therapeutic potential. British Society for Antimicrobial Chemotherapy BSAC-2018-0095; NC3Rs PhD Studentship NC/V001582/1; Biotechnology and Biological Sciences Research Council New Investigator Award BB/V007823/1; Academy of Medical Sciences/the Wellcome Trust/the Government Department of Business, Energy and Industrial Strategy/the Bri- tish Heart Foundation/Diabetes UK Springboard Award [SBF006\1040; Research Grant BB/T007168/1 from the Biotechnology and Biological Sciences Research Council.

The use of polymers in the development of biomaterials for various biomedical applications has become increasingly important in recent decades. To match the innate properties of biological tissues, the polymer-based tissue scaffolds must have the desired structural and functional properties. However, the polymer-based hydrogels prepared by conventional methods are often delicate and fragile and require pre-stabilisation. This necessitates the exploration of bio-friendly cross-linkers that promote kinetic or reversible crosslinking in the polymer network of hydrogels and must be nontoxic to cells and tissues. The light initiators with well-organized multiphoton cross sections that are reactive at specific wavelengths could be ideal candidates. This chapter reviews the fabrication of solid or viscoelastic biological scaffolds by multiphoton lithography (MPL) of liquids. It describes the similarities and differences between conventional and MPL photo polymerization of biological scaffolds in terms of synthesis chemistry, properties, and their relevance to biological applications. These photosensitive scaffolds could be useful biomaterials for their biomedical applications.

Background The current mutagenesis tools for Acinetobacter baumannii leave selection markers or residual sequences behind, or involve tedious counterselection and screening steps. Furthermore, they are usually adapted for model strains, rather than for MDR clinical isolates. Objectives To develop a scar-free genome-editing tool suitable for chromosomal and plasmid modifications in MDR A. baumannii AB5075. Methods We prove the efficiency of our adapted genome-editing system by deleting the multidrug efflux pumps craA, cmlA5 and resistance island 2 (RI2), as well as curing plasmid p1AB5075, and combining these mutations. We then characterized the susceptibility of the mutants compared with the WT to different antibiotics (i.e. chloramphenicol, amikacin and tobramycin) by disc diffusion assays and determined the MIC for each strain. Results We successfully adapted the genome-editing protocol to A. baumannii AB5075, achieving a double recombination frequency close to 100% and routinely securing the construction of a mutant within 10 working days. Furthermore, we show that both CraA and p1AB5075 are involved in chloramphenicol resistance, and that RI2 and p1AB5075 play a role in resistance to amikacin and tobramycin. Conclusions We have developed a versatile and highly efficient genome-editing tool for A. baumannii. We have demonstrated it can be used to modify both the chromosome and native plasmids. By challenging the method, we show the role of CraA and p1AB5075 in antibiotic resistance.

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival ofAntibiotics, like penicillin, are the foundation of modern medicine, but bacteria are evolving to resist their effects. Some of the most harmful pathogens belong to a group called the 'Gram-negative bacteria', which have an outer layer – called the cell envelope – that acts as a drug barrier. This envelope contains antibiotic resistance proteins that can deactivate or repel antibiotics or even pump them out of the cell once they get in. One way to tackle antibiotic resistance could be to stop these proteins from working. Proteins are long chains of building blocks called amino acids that fold into specific shapes. In order for a protein to perform its role correctly, it must fold in the right way. In bacteria, a protein called DsbA helps other proteins fold correctly by holding them in place and inserting links called disulfide bonds. It was unclear whether DsbA plays a role in the folding of antibiotic resistance proteins, but if it did, it might open up new ways to treat antibiotic resistant infections. To find out more, Furniss, Kaderabkova et al. collected the genes that code for several antibiotic resistance proteins and put them into

The discovery of penicillin over 90 years ago and its subsequent uptake by healthcare systems around the world revolutionised global health. It marked the beginning of a golden age in antibiotic discovery with new antibiotics readily discovered from natural sources and refined into therapies that saved millions of lives. Towards the end of the last century, the rate of discovery slowed to a near standstill. The lack of discovery is compounded by the rapid emergence and spread of bacterial pathogens that exhibit resistance to multiple antibiotic therapies and threaten the sustainability of global healthcare systems. Acinetobacter baumannii is an opportunistic pathogen whose prevalence and impact has grown significantly over the last 20 years. It is recognised as a barometer of the antibiotic resistance crisis due to the diverse array of mechanisms by which it can become resistant.

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug- resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.IMPACT STATEMENTDisruption of disulfide bond formation sensitizes resistant Gram- negative bacteria expressing β-lactamases and mobile colistin resistance enzymes to currently available antibiotics.

The Brunel Research Innovation and Enterprise fund; British Council/Newton Fund; National Natural Science Foundation of China; China Postdoctoral Science Foundation; Fundamental Research Funds for Central University; Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry, Changchun Institue of Applied Chemistry, Chinese Academy of Sciences

23 Págs. Pictures., The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally., The animal study was funded under an Innovate UK and Genus funded sub-contract from the Roslin Institute and APHA (CSKN0019). This research was also financially supported by the Department for Environment, Food and Rural Affairs (DEFRA), the Scottish Government and the Welsh Government. The development and refinement of the scoring systems was supported by the Swedish Environmental Protection Agency

Burn wounds can create significant damage to human skin, compromising one of the key barriers to infection. The leading cause of death among burn wound patients is infection. Even in the patients that survive, infections can be notoriously difficult to treat and can cause lasting damage, with delayed healing and prolonged hospital stays. Biofilm formation in the burn wound site is a major contributing factor to the failure of burn treatment regimens and mortality as a result of burn wound infection. Bacteria forming a biofilm or a bacterial community encased in a polysaccharide matrix are more resistant to disinfection, the rigors of the host immune system, and critically, more tolerant to antibiotics. Burn wound-associated biofilms are also thought to act as a launchpad for bacteria to establish deeper, systemic infection and ultimately bacteremia and sepsis. In this review, we discuss some of the leading burn wound pathogens and outline how they regulate biofilm formation in the burn wound microenvironment. We also discuss the new and emerging models that are available to study burn wound biofilm formation in vivo. Funding Agencies|British Society for Antimicrobial Chemotherapy [BSAC-2018-0095]; Innovate UK Smart Grant [37800]; FRAME, Young European Research University Network Mobility Award, NC3Rs PhD Studentship [NC/V001582/1]; BBSRC New Investigator AwardUK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC) [BB/V007823/1]; Academy of Medical Sciences/the Wellcome Trust/ the Government Department of Business, Energy and Industrial Strategy/the British Heart Foundation/Diabetes UK Springboard Award [SBF006\1040]

The discovery of penicillin over 90 years ago and its subsequent uptake by healthcare systems around the world revolutionised global health. It marked the beginning of a golden age in antibiotic discovery with new antibiotics readily discovered from natural sources and refined into therapies that saved millions of lives. Towards the end of the last century, the rate of discovery slowed to a near standstill. The lack of discovery is compounded by the rapid emergence and spread of bacterial pathogens that exhibit resistance to multiple antibiotic therapies and threaten the sustainability of global healthcare systems. Acinetobacter baumannii is an opportunistic pathogen whose prevalence and impact has grown significantly over the last 20 years. It is recognised as a barometer of the antibiotic resistance crisis due to the diverse array of mechanisms by which it can become resistant.

Copyright © 2020 The Author(s). African swine fever virus (ASFV) causes a lethal, haemorrhagic disease in domestic swine that threatens pig production across the globe. Unlike domestic pigs, warthogs, which are wildlife hosts of the virus, do not succumb to the lethal effects of infection. There are three amino acid differences between the sequence of the warthog and domestic pig RELA protein; a subunit of the NF-κB transcription factor that plays a key role in regulating the immune response to infections. Domestic pigs with all 3 or 2 of the amino acids from the warthog RELA orthologue have been generated by gene editing. To assess if these variations confer resilience to ASF we established an intranasal challenge model with a moderately virulent ASFV. No difference in clinical, virological or pathological parameters were observed in domestic pigs with the 2 amino acid substitution. Domestic pigs with all 3 amino acids found in warthog RELA were not resilient to ASF but a delay in onset of clinical signs and less viral DNA in blood samples and nasal secretions was observed in some animals. Inclusion of these and additional warthog genetic traits into domestic pigs may be one way to assist in combating the devastating impact of ASFV. This work was funded by InnovateUK Crop and Livestock Disease Challenges: Validation of Genome Edited Disease Resistant Pig grant BB/M028313/1 and by Genus PLC. Work at the APHA was completed under an InnovateUK and Genus funded sub-contract, CSKN0019. In addition the work was in part supported by BBSRC ISPG funding BBS/E/D/10002071, BBS/E/D/20002172, BBS/E/D/20002174, BB/P013740/1 and BB/P013759/1 to the Roslin Institute.

Rapid and effective virus inactivation is an essential step for safe diagnostic testing and for research and vaccine development using infectious viruses. We characterised the reduction of African Swine Fever Virus (ASFV) infectivity using Virkon™ S (Lanxess) 1% w/v disinfectant, FACS™ Lysing buffer (BD), and AVL™ buffer (Qiagen), using porcine cell culture. No virus was detected following a 30 s 20:1 v/v mixing ratio of Virkon™ S 1% with high titre ASFV, supporting its effective use as a laboratory surface disinfectant. FACS™ Lysing and AVL™ buffers also inactivated ASFV, permitting safe removal of treated infected samples from high containment facilities.

Meeting report Area of expertise: My research interests focus on identifying novel compounds that can disrupt key phenotypes associated with antibiotic resistance such as biofilm formation and motility in the pathogens Acinetobacter baumannii and Pseudomonas aeruginosa. To this end we have developed a range of screens and assays to facilitate compound identification and mechanism of action profiling in these pathogens. These include: Antibiotic Resistance Inhibitor Screens, Biofilm Inhibition Screens, Biofilm Disruption Screens, Signalling Pathway Specific Biosensor Screens , Mechanism of Action Assays, Resistance Development Screens, Antibiotic Resistance Inhibitor Screens, In vivo biofilm and pathogenicity models to validate lead compounds and perform toxicity profiling. Future goals: Future goals include being part of a collaborative multidisciplinary drug discovery network that has access to diverse and bespoke extract/compound collections and the expertise to identify the bioactive leads, characterise their mechanism of action and ultimately take them forward to clinical trials. Further to this the goal is to target national and international funding opportunities that will facilitate taking active compounds right through the drug discovery pipeline from the bench to the bedside. I am hoping to identify collaborators across all aspects of drugs discovery, particularly collaborators with: Access to compound or extract collections, Expertise in compound identification ,Clinical trial expertise.

Classical Swine Fever Virus (CSFV) is an ongoing threat to the pig industry due to its high transmission and mortality rates associated with infection. Live attenuated vaccines such as the CSFV C strain vaccine are capable of protecting against infection within 5 days of vaccination, but the molecular mechanisms through which this early protection is mediated have yet to be established. In this study, we compared the response of pigs vaccinated with the C strain to non-vaccinated pigs both challenged with a pathogenic strain of CSFV. Analysis of transcriptomic data from the tonsils of these animals during the early stages after vaccination and challenge reveals a set of regulated genes that appear throughout the analysis. Many of these are linked to the ISG15 antiviral pathway suggesting it plays a key role in the rapid and early protection conferred by C strain vaccination.

© 2019 The Authors. The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di‐GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c‐di‐GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c‐di‐GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c‐di‐GMP being a key second messenger that silences energy‐costing systems during early colonization phase and biofilm formation, while low c‐di‐GMP levels unleash T6SS and T4SS to advance plant colonization. Biotechnology and Biological Sciences Research Council. Grant Numbers: BB/L007959/1, BB/M02735X/1 Ministry of Science and Technology, Taiwan. Grant Number: 104-2311-B-001-025-MY3

Diet has a significant impact on shaping the human gut microbiome; this could be partially due to the ability of certain phytochemicals found in the diet to interfere with native microbial signalling. Compounds isolated from foods such as garlic, coffee and grapefruit have all been shown to have a significant impact on the virulence potential of known pathogens such as Pseudomonas aeruginosa in vitro. This impact on pathogenicity is the result of the ability of phytochemicals isolated from these foods to inhibit of quorum sensing. These phytochemicals with quorum sensing inhibitory activity have the potential to be developed as next generation antimicrobials, which inhibit key virulence processes utilised by bacteria to establish infection while reducing the probability of selecting for resistance. These phytochemicals have the potential to be exploited as ingredients in nutraceuticals, functional food ingredients and pharmaceuticals that are capable of improving the efficacy of traditional antibiotic therapies.

Summary In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries. A combination of unique physicochemical properties and spatial niche‐specific substrates, in wide‐ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel bioactivities. One such area of ongoing research is the discovery of compounds that interfere with the cell–cell signalling process called quorum sensing (QS). Described as the next generation of antimicrobials, these compounds can target virulence and persistence of clinically relevant pathogens, independent of any growth‐limiting effects. Marine sponges are a rich source of microbial diversity, with dynamic populations in a symbiotic relationship. In this study, we have harnessed the QS inhibition (QSI) potential of marine sponge microbiota and through culture‐based discovery have uncovered small molecule signal mimics that neutralize virulence phenotypes in clinical pathogens. This study describes for the first time a marine sponge Psychrobacter sp. isolate B98C22 that blocks QS signalling, while also reporting dual QS/QSI activity in the Pseudoalteromonas sp. J10 and Paracoccus JM45. Isolation of novel QSI activities has significant potential for future therapeutic development, of particular relevance in the light of the pending perfect storm of antibiotic resistance meeting antibiotic drug discovery decline.

Bacteria produce toxins to enhance their competitiveness in the colonization of an environment as well as during an infection. The delivery of toxins into target cells is mediated by several types of secretion systems, among them our focus is Type III and Type VI Secretion Systems (T3SS and T6SS, respectively). A thorough methodology is provided detailing how to identify if cyclic di-GMP signaling plays a role in the P. aeruginosa toxin delivery mediated by T3SS or T6SS. This includes in vitro preparation of the samples for Western blot analysis aiming at detecting possible c-di-GMP-dependent T3SS/T6SS switch, as well as in vivo analysis using the model organism Galleria mellonella to demonstrate the ecological and pathogenic consequence of the switch between these two secretion systems.

Bacteria produce toxins to enhance their competitiveness in the colonization of an environment as well as during an infection. The delivery of toxins into target cells is mediated by several types of secretion systems, among them our focus is Type III and Type VI Secretion Systems (T3SS and T6SS, respectively). A thorough methodology is provided detailing how to identify if cyclic di-GMP signaling plays a role in the P. aeruginosa toxin delivery mediated by T3SS or T6SS. This includes in vitro preparation of the samples for Western blot analysis aiming at detecting possible c-di-GMP-dependent T3SS/T6SS switch, as well as in vivo analysis using the model organism Galleria mellonella to demonstrate the ecological and pathogenic consequence of the switch between these two secretion systems.

Additive manufacturing or 3D printing has spearheaded a revolution in the biomedical sector allowing the rapid prototyping of medical devices. The recent advancements in bioprinting technology are enabling the development of potential new therapeutic options with respect to tissue engineering and regenerative medicines. Bacterial polysaccharides have been shown to be a central component of the inks used in a variety of bioprinting processes influencing their key features such as the mechanical and thermal properties, printability, biocompatibility, and biodegradability. However, the implantation of any foreign structure in the body comes with an increased risk of bacterial infection and immunogenicity. In recent years, this risk is being potentiated by the rise in nosocomial multidrug-resistant bacterial infections. Inks used in bioprinting are being augmented with antimicrobials to mitigate this risk. The applications of bacterial polysaccharide-based bioinks have the potential to act as a key battlefront in the war against antibiotic resistance. This paper reviews the range of bacterial polysaccharides used in bioprinting and discusses the potential of various bioactive polysaccharides to be integrated into these inks.

The rapid unchecked rise in antibiotic resistance over the last few decades has led to an increased focus on the need for alternative therapeutic strategies for the treatment and clinical management of microbial infections. In particular, small molecules that can suppress microbial virulence systems independent of any impact on growth are receiving increased attention. Quorum sensing (QS) is a cell-to-cell signalling communication system that controls the virulence behaviour of a broad spectrum of bacterial pathogens. QS systems have been proposed as an effective target, particularly as they control biofilm formation in pathogens, a key driver of antibiotic ineffectiveness. In this study, we identified coumarin, a natural plant phenolic compound, as a novel QS inhibitor, with potent anti-virulence activity in a broad spectrum of pathogens. Using a range of biosensor systems, coumarin was active against short, medium and long chain N-acyl-homoserine lactones, independent of any effect on growth. To determine if this suppression was linked to anti-virulence activity, key virulence systems were studied in the nosocomial pathogen Pseudomonas aeruginosa. Consistent with suppression of QS, coumarin inhibited biofilm, the production of phenazines and swarming motility in this organism potentially linked to reduced expression of the rhlI and pqsA quorum sensing genes. Furthermore, coumarin significantly inhibited biofilm formation and protease activity in other bacterial pathogens and inhibited bioluminescence in Aliivibrio fischeri. In light of these findings, coumarin would appear to have potential as a novel quorum sensing inhibitor with a broad spectrum of action.

LysR-type transcriptional regulators (LTTRs) are the most common family of transcriptional regulators found in the opportunistic pathogenPseudomonas aeruginosa. They are known to regulate a wide variety of virulence determinants and have emerged recently as positive global regulators of pathogenicity in a broad spectrum of important bacterial pathogens. However, in spite of their key role in modulating expression of key virulence determinants underpinning pathogenic traits associated with the process of infection, surprisingly few are found to be transcriptionally altered by contact with host cells. BvlR (PA14_26880) an LTTR of previously unknown function, has been shown to be induced in response to host cell contact, and was therefore investigated for its potential role in virulence. BvlR expression was found to play a pivotal role in the regulation of acute virulence determinants such as type III secretion system and exotoxin A production. BvlR also played a key role inP. aeruginosapathogenicity within theCaenorhabditis elegansacute model of infection. Loss of BvlR led to an inability to form tight microcolonies, a key step in biofilm formation in the cystic fibrosis lung, although surface attachment was increased. Unusually for LTTRs, BvlR was shown to exert its influence through the transcriptional repression of many genes, including the virulence-associatedcupAandalggenes. This highlights the importance of BvlR as a new virulence regulator inP. aeruginosawith a central role in modulating key events in the pathogen–host interactome.

The glutathione-binding protein of Mytilus edulis possesses only one tryptophan per polypeptide. Quenching of intrinsic fluorescence due to this residue was studied in the presence of glutathione S-transferase ligands; hematin, bilirubin, biliverdin, bromosulphophthalein, 1-anilino-8-naphthalene sulphonate, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and sodium deoxycholate as well as in the presence of triethyltin bromide. Kd values were estimated from these experiments and were found to be 38–310 μM. Based on non-denaturing electrophoresis, the protein was found to have a native molecular weight of 50 kDa. Taken together with previously reported subunit molecular weights in the region of 25 kDa, this indicates that this protein has a dimeric quaternary structure.

PyeR (PA4354) is a novel member of the ArsR family of transcriptional regulators and modulates biofilm formation in Pseudomonas aeruginosa. Characterization of this regulator showed that it has negative autoregulatory properties and binds to a palindromic motif conserved among PyeR orthologues. These characteristics are in line with classical ArsR-family regulators, as is the fact that PyeR is part of an operon structure (pyeR-pyeM-xenB). However, PyeR also exhibits some atypical features in comparison with classical members of the ArsR family, as it does not harbour metal-binding motifs and does not appear to be involved in metal perception or resistance. Hence, PyeR belongs to a subgroup of non-classical ArsR-family regulators and is the second ArsR regulator shown to be involved in biofilm formation.

Rationale: Chronic bacterial infection leading to an irreversible decline in lung function is the main cause of mortality and morbidity in people with cystic fibrosis (CF). Anaerobic bacteria have been recently shown to be present in sputum in high numbers in CF; however their role in infection and inflammation needs further investigation. Short chain fatty acids (SCFA) are known to be produced as metabolic by-products of anaerobic bacteria and we investigated if their role in initiating the pro-inflammatory cascade. Objectives: To investigate the role SCFAs play in the CF lung and if they are pro-inflammatory mediators. Methods and Results: We investigated the protein and mRNA expression levels of GPR41 and GPR 43, G protein coupled receptors for SCFAs, using Western blot analysis and reverse transcriptase-polymerase chain reaction. We found that GPR41 and GPR 43 protein and mRNA are highly expressed in cystic fibrosis bronchial epithelial cells, (CFBE 41o) as compared with normal human bronchial epithelial cells (16HBE14o) and this was shown to be significant (p< 0.001). We evaluated the role of IL-8 production by quantifying the most known prevalent SCFA, sodium butyrate on CFBE 41o and 16HBE14o using enzyme-linked-linked immunosorbent assay (ELISA). We found increased IL-8 production in CFBE cells compared to HBE cells. Conclusion: We found differentiated increased expression of GPR 41 in CFBE 41o cells compared with 16HBE14o. We also found that sodium butyrate, a SCFA, increases IL-8 production in CFBE cells.