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Biomedical subjects

Paul Williams

Publications and source records attributed to Paul Williams.

At least 19 recordsLinked to original sources

Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity.

The main source of Leishmania infantum infection in humans is a naturally infected dog. This study reports on the infectivity to phlebotomine sandflies (Lutzomyia longipalpis) of serologically positive mongrel dogs that differed in clinical status, haematology and humoral responses to immunoglobulin (Ig) G(T) (total anti-Leishmania IgG), IgG(1) and IgG(2) subclasses of antibody to crude antigen of L. infantum. Forty-five female L. longipalpis were allowed to feed directly on the ears of dogs classified as asymptomatic, oligosymptomatic or symptomatic before being dissected five days later. Promastigotes were detected in 88% of the dissected sandflies. The highest rate of infectivity to sandflies was found in symptomatic dogs, followed by oligosymptomatic and asymptomatic animals. The results suggest that dogs naturally infected with L. infantum with higher total IgG and IgG(2) concentrations and lower haematocrit levels were able to infect the highest proportion of L. longipalpis. No correlation was observed between anaemia and the intensity of clinical signs. Symptomatic dogs presented the highest infection rate and intensity of infection.

Animals↗

NAD(P)H oxidases regulate HIF-2alpha protein expression.

Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary and sporadic renal cell carcinoma (RCC). In the absence of VHL, the alpha subunits of heterodimeric hypoxia-inducible transcription factors (HIF-1alpha and HIF-2alpha) are stabilized. Reactive oxygen species, generated by NAD(P)H oxidases, are involved in signaling cascades of malignant growth. We show that in VHL-deficient cells p22phox, Nox4 protein levels and NADPH-dependent superoxide generation are increased. Reintroduction of VHL into the VHL-deficient cells down-regulates the expression of p22phox and NADPH-dependent superoxide generation. Inhibition of the 26 S proteasome in VHL-expressing cells increased p22phox protein levels, which correlated with an increase of NADPH-dependent superoxide generation. We also show that p22phox co-immunoprecipitates with VHL in vivo. Moreover, p22phox is a target of ubiquitination. Importantly, in VHL-deficient cells, diphenyleneiodonium chloride (DPI), an inhibitor of Nox oxidases, decreased the expression of HIF-2alpha. Down-regulation of Nox1, Nox4, and p22phox expression by small interfering RNA also decreased HIF-2alpha protein expression and inhibited Akt and 4E-BP1 phosphorylation, suggesting that a translational mechanism is involved in maintaining HIF-2alpha in VHL-deficient cells. Colony formation by RCC 786-O in soft agar was markedly inhibited by DPI. Moreover, DPI significantly inhibited RCC 786-O tumor formation in athymic mice. Collectively, the data demonstrate that VHL protein exerts its tumor suppressor action, at least partially, via inhibition of p22phox-based Nox4/Nox1 NADPH oxidase-dependent reactive oxygen species generation.

Basic Helix-Loop-Helix Proteins↗

Use of the sustained pain-free plus no adverse events endpoint in clinical trials of triptans in acute migraine.

OBJECTIVE: To assess the relationship between the sustained pain free (SPF) and adverse event (AE) rates associated with six oral serotonin 5-HT(1B/1D) receptor agonists (triptans) used for the treatment of acute migraine, employing data from a previous meta-analysis (that included almotriptan, eletriptan, naratriptan, rizatriptan, sumatriptan and zolmitriptan, but not frovatriptan) in order to provide a rationale for the use of the rate of patients who are SPF plus no AE (SNAE) as an endpoint in trials of medications for acute migraine. BACKGROUND: The attributes of drug treatment for acute migraine that are most important to people who experience them are complete pain relief, lack of recurrence, rapid onset and lack of AEs. The endpoints used to assess therapy for acute migraine do not always address these elements. METHODS: The relationship between SPF and AE rates was explored using nonparametric regression techniques, and the box-plot method was used to identify outliers. The estimated SNAE rate for each triptan was calculated with and without assuming independence between efficacy and tolerability. RESULTS: At the level of the individual agent, there was a significant relationship between the efficacy and tolerability of each triptan, with the exception of almotriptan 12.5 mg, which had an AE rate approximately 30% lower than would be expected, and eletriptan 20 mg, which had an AE rate approximately 20% higher than would be expected, on the basis of their efficacy. Almotriptan 12.5 mg and eletriptan 20 mg had the highest and lowest base-case values for SNAE, respectively, and both qualified statistically as outliers to the distribution of SNAE values obtained with the other triptans. The probability that each triptan was superior to the reference agent (sumatriptan 100 mg) in terms of SNAE was calculated across all possible values (at the level of the individual patient) for the relationship between efficacy and tolerability. Again, almotriptan 12.5 mg and eletriptan 20 mg had the highest and lowest values for their SNAE rates, respectively, and almotriptan 12.5 mg qualified statistically as an outlier from the distribution of the probabilities obtained for the other triptans. CONCLUSION: This analysis determined that higher SPF rates were strongly associated with higher AE rates, with the notable exception of almotriptan 12.5 mg, which had a lower than expected AE rate, resulting in the highest SNAE rate of the included triptans and doses. SNAE is a useful measure that can be used to discriminate between therapies and this endpoint incorporates the attributes that are most relevant to patient satisfaction with treatment. We recommend calculation of the SNAE rate at the level of the individual patient in future clinical trials of medication for the treatment of acute migraine to facilitate selection of a treatment for acute migraine that offers the best chance for effective management.

Acute Disease↗

N-acyl homoserine lactones are degraded via an amidolytic activity in Comamonas sp. strain D1.

Comamonas strain D1 enzymatically inactivates quorum-sensing (QS) signal molecules of the N-acyl homoserine lactone (N-AHSL) family, and exhibits the broadest inactivation range of known bacteria. It degrades N-AHSL with acyl-side chains ranging from 4 to 16 carbons, with or without 3-oxo or 3-hydroxy substitutions. N-AHSL degradation yields HSL but not N-acyl homoserine: strain D1 therefore harbors an amidohydrolase activity. Strain D1 is the fifth bacterium species in which an N-AHSL amidohydrolase is described. Consistent with its N-AHSL degradation ability, strain D1 efficiently quenches various QS-dependent functions in other bacteria, such as violacein production by Chromobacterium violaceum and pathogenicity and antibiotic production in Pectobacterium.

4-Butyrolactone↗

Comprehensive profiling of N-acylhomoserine lactones produced by Yersinia pseudotuberculosis using liquid chromatography coupled to hybrid quadrupole-linear ion trap mass spectrometry.

A method for the comprehensive profiling of the N-acylhomoserine lactone (AHL) family of bacterial quorum-sensing molecules is presented using liquid chromatography (LC) coupled to hybrid quadrupole-linear ion trap (QqQLIT) mass spectrometry. Information-dependent acquisition (IDA), using triggered combinations of triple-quadrupole and linear ion trap modes in the same LC-MS/MS run, was used to simultaneously screen, quantify and identify multiple AHLs in a single sample. This MS method uses common AHL fragment ions attributed to the homoserine moiety and the 3-oxo-, 3-hydroxy- or unsubstituted acyl side chains, to identify unknown AHLs in cell-free culture supernatants in an unbiased manner. This LC-MS technique was applied to determine the relative molar ratios of AHLs produced by Yersinia pseudotuberculosis and the consequences of inactivating by mutation either or both of the AHL synthase genes (ypsI and ytbI) on AHL profile and concentration. The Y. pseudotuberculosis wild type but not the ypsI ytbI double mutant produced at least 24 different AHLs with acyl chains ranging from C4 to C15 with or without 3-oxo or 3-hydroxy substituents. YtbI, in contrast to YpsI, could direct the synthesis of all of the AHLs identified. The most abundant and hence most biologically relevant Y. pseudotuberculosis AHLs were found to be the 3-oxo-substituted C6, C7 and C8 AHLs and the unsubstituted C6 and C8 compounds. The LC-QqQLIT methodology is broadly applicable to quorum-sensing signal molecule analysis and can provide comprehensive AHL profiles and concentrations from a single sample and simultaneously collect confirmatory spectra for each AHL identified.

4-Butyrolactone↗

Persistent neurobehavioral effects of early postnatal domoic acid exposure in rats.

Domoic acid (DA) is a marine biotoxin, produced by the diatom Pseudo-nitzchia spp., which has been shown to cause cognitive impairment in adults who are exposed via contaminated seafood. The neurobehavioral consequences of developmental exposure are much less well understood. In a previous study, we showed that a single prenatal exposure to DA in rats at mid-gestation caused neurobehavioral changes that persist into adulthood including increased susceptibility to the benchmark amnestic drug scopolamine. In the current study, we examined the lasting neurobehavioral consequences of DA exposure on the first day of postnatal life, a time in rats marking the completion of the major phase of neuroproliferation and corresponding to week 24 of human gestation. The effects of DA exposure at doses from 0.025-0.1 mg/kg (s.c.) twice per day on each of postnatal days 1 and 2 were compared with vehicle-treated controls and rats treated by the same protocol with 1 mg/kg of kainic acid. Following kainic acid exposure, a sex-selective effect was seen with females but not males showing a significant slowing of response latency in the radial-arm maze. The high DA dose of 0.1 mg/kg was quite toxic causing lethality in all of the offspring exposed and this group was excluded from further analysis. When the offspring in the 0.05 mg/kg DA dose group were tested, significant hypoactivity in the Figure-8 maze was observed during adolescence. No significant DA effects were seen in response latency or choice accuracy on the radial-arm maze during either acquisition or with challenge of the amnestic drug scopolamine. Early postnatal DA exposure in the rat can be lethal and sublethal exposure can cause neurobehavioral effects manifest in modest hypoactivity during the adolescent period. However, the sublethal persistent neurobehavioral toxicity appears to be less pervasive than reported effects following DA administered mid-gestation.

Animals↗

Foot-and-mouth disease - quantification and size distribution of airborne particles emitted by healthy and infected pigs.

There is strong evidence to suggest that foot-and-mouth disease (FMD) can be transmitted by airborne virus up to many kilometres from a virus source. Atmospheric dispersion models are often used to predict where this disease might spread. This study investigated whether FMD virus (FMDV) aerosol has specific characteristics which need to be taken into consideration in these models. The characteristics and infectiousness of particles emitted by 12 pigs have been studied pre- and post-infection with O UKG 2001 FMDV. Aerosol generated by individual pigs was found log normally distributed in the range 0.015-20.0microm with concentrations between 1000 and 10000cm(-3) at the smallest size and <1cm(-3) above 10microm. No differences in either the total number of particles produced or their size distribution were detected between uninfected and infected pigs. However, a correlation between aerosol concentration and animal activity was found with a more active pig producing significantly greater concentrations than those that were less active. Viable virus was found up to a maximum of 6.3 log TCID(50)/24h/animal. The virus was distributed almost equally across the three size ranges; <3, 3-6 and >6microm. No correlation could be established between the production of virus and animal activity. In general the production of airborne virus closely followed the detection of viraemia in the blood and the presence of clinical symptoms. However, in one instance a pig excreted as much airborne virus as the other animals in the study, but with less virus detected in its blood. The results suggest that there is little merit in including a sophisticated virus release pattern based on physical activity periods or FMDV aerosol size spectrum, together with the appropriate dry deposition calculations, in models used to predict airborne spread of FMD. An estimate of the total daily virus production based on the clinical assessment of disease and virus strain is sufficient as input.

Air Microbiology↗

Regulatory roles of spnT, a novel gene located within transposon TnTIR.

The transposon TnTIR contains spnIR quorum-sensing system regulating sliding motility and the production of nuclease, biosurfactant, and prodigiosin in Serratia marcescens. Within TnTIR, a gene named spnT is upstream of and co-transcribed with spnI. SpnT is a cytoplasmic protein and its level peaks during early stationary phase. spnT over-expression resulted in inhibition of sliding motility and synthesis of prodigiosin, and biosurfactant similar to spnR. spnT but not spnR over-expression induced cell elongation and aberrant DNA replication in S. marcescens and Escherichia coli strains. In comparison with wild-type E. coli strain, over-expression of spnT in an E. coli priA and dnaC double-mutant strain did not lead to the aberrant cell morphology phenotypes, suggesting SpnT may act through the recombination-dependent DNA replication system. As spnT over-expression inhibited swarming but not swimming motility, SpnT may indirectly function as a negative regulator of surface-dependent migration and secondary metabolite production.

Bacterial Proteins↗

Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover.

HD-GYP is a protein domain of unknown biochemical function implicated in bacterial signaling and regulation. In the plant pathogen Xanthomonas campestris pv. campestris, the synthesis of virulence factors and dispersal of biofilms are positively controlled by a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC and by cell-cell signaling mediated by the diffusible signal molecule DSF (diffusible signal factor). The RpfG/RpfC two-component system has been implicated in DSF perception and signal transduction. Here we show that the role of RpfG is to degrade the unusual nucleotide cyclic di-GMP, an activity associated with the HD-GYP domain. Mutation of the conserved H and D residues of the isolated HD-GYP domain resulted in loss of both the enzymatic activity against cyclic di-GMP and the regulatory activity in virulence factor synthesis. Two other protein domains, GGDEF and EAL, are already implicated in the synthesis and degradation respectively of cyclic di-GMP. As with GGDEF and EAL domains, the HD-GYP domain is widely distributed in free-living bacteria and occurs in plant and animal pathogens, as well as beneficial symbionts and organisms associated with a range of environmental niches. Identification of the role of the HD-GYP domain thus increases our understanding of a signaling network whose importance to the lifestyle of diverse bacteria is now emerging.

Amino Acid Sequence↗

Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment.

The first choice of treatment for American cutaneous leishmaniasis is the pentavalent antimonial drug. Although it has been shown that this treatment is mostly effective and indicated, some disadvantages should be taken into account such as side effects, long term treatment inconveniences and counter-indication for patients suffering from cardiopathy, nephropathy; yet, aging, pregnancy and other conditions. With the advent of the vaccine anti-American cutaneous leishmaniasis as a prophylactic measure, studies on therapy using the vaccine associated or not with other drugs have been performed by many investigators and it is currently among the alternative treatments and prevention measures for American cutaneous leishmaniasis. In conclusion, the association between antimony and vaccine (immunochemotherapy) showed the same cure rate when compared with the standard treatment (100%) and it was also able to reduce the salt volume in 17.9% and treatment length from 87 to 62 days, decreasing side effects.

Adolescent↗

Quorum sensing.

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4-Butyrolactone↗

4-quinolone signalling in Pseudomonas aeruginosa: old molecules, new perspectives.

In Pseudomonas aeruginosa, diverse virulence determinants and secondary metabolites are regulated via the action of a hierarchical quorum-sensing system which integrates two chemically distinct classes of signal molecules, the N-acylhomoserine lactones (AHLs) and the 4-quinolones (4Qs). Synthesis of the pseudomonas quinolone signal, 2-heptyl-3-hydroxy-4-quinolone (PQS) depends on the pqsABCDE locus which is responsible for generating multiple 4Qs including 2-heptyl-4-quinolone (HHQ), the immediate PQS precursor. Exported HHQ is taken up by adjacent bacterial cells and converted into PQS by PqsH, a putative mono-oxygenase. In addition, PQS regulates its own production by driving the expression of pqsABCDE through a direct interaction with PqsR (MvfR). PQS regulates diverse target genes including those coding for elastase, rhamnolipid, the PA-IL lectin and pyocyanin via the action of PqsE as well as influencing biofilm development and impacting on cellular fitness. Furthermore, 4Q signalling is not restricted to P. aeruginosa raising the possibility of cross-talk with other related bacterial species which occupy similar ecological niches.

4-Quinolones↗

The 1.6A X-ray structure of the unusual c-type cytochrome, cytochrome cL, from the methylotrophic bacterium Methylobacterium extorquens.

The structure of cytochrome cL from Methylobacterium extorquens has been determined by X-ray crystallography to a resolution of 1.6 A. This unusually large, acidic cytochrome is the physiological electron acceptor for the quinoprotein methanol dehydrogenase in the periplasm of methylotrophic bacteria. Its amino acid sequence is completely different from that of other cytochromes but its X-ray structure reveals a core that is typical of class I cytochromes c, having alpha-helices folded into a compact structure enclosing the single haem c prosthetic group and leaving one edge of the haem exposed. The haem is bound through thioether bonds to Cys65 and Cys68, and the fifth ligand to the haem iron is provided by His69. Remarkably, the sixth ligand is provided by His112, and not by Met109, which had been shown to be the sixth ligand in solution. Cytochrome cL is unusual in having a disulphide bridge that tethers the long C-terminal extension to the body of the structure. The crystal structure reveals that, close to the inner haem propionate, there is tightly bound calcium ion that is likely to be involved in stabilization of the redox potential, and that may be important in the flow of electrons from reduced pyrroloquinoline quinone in methanol dehydrogenase to the haem of cytochrome cL. As predicted, both haem propionates are exposed to solvent, accounting for the unusual influence of pH on the redox potential of this cytochrome.

Amino Acid Sequence↗

A Study for a retrieved implant of ceramic-on-ceramic total hip arthroplasty.

A Mittelmeier-type ceramic on ceramic total hip arthroplasty (THA) was retrieved because of aseptic loosening 17 years after implantation. The extent of wear was assessed by scanning electron microscopy. The synovial fluid and the tissue surrounding the implant were also examined. Scanning electron microscopy analysis of the ball head showed that the main wear zone had grade IV wear. The stripe wear zones showed grain pullout regions (grade 5 wear). Pathologically, chronic inflammation was observed in the surrounding tissue. The debris particles in the synovial fluid were polygonal and approximately 3 x 4 microm. Thus, the Mittelmeier-type THA proved excellent wear resistance. Further longevity of ceramic on ceramic THA may indeed become a reality with improvement of the design and quality of alumina.

Arthroplasty, Replacement, Hip↗

Functional genetic analysis reveals a 2-Alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria.

Pseudomonas aeruginosa synthesizes diverse 2-alkyl-4(1H)-quinolones (AHQs), including the signaling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), via the pqsABCDE locus. By examining the genome databases, homologs of the pqs genes were identified in other bacteria. However, apart from P. aeruginosa, only Burkholderia pseudomallei and B. thailandensis contained a complete pqsA-E operon (termed hhqA-E). By introducing the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants, we show that they are functionally conserved and restore virulence factor and PQS production. B. pseudomallei, B. thailandensis, B. cenocepacia, and P. putida each produced 2-heptyl-4(1H)-quinolone (HHQ), but not PQS. Mutation of hhqA in B. pseudomallei resulted in the loss of AHQ production, altered colony morphology, and enhanced elastase production, which was reduced to parental levels by exogenous HHQ. These data reveal a role for AHQs in bacterial cell-to-cell communication beyond that seen in P. aeruginosa.

Burkholderia pseudomallei↗

Long- and short-chain plant-produced bacterial N-acyl-homoserine lactones become components of phyllosphere, rhizosphere, and soil.

Two N-acyl-homoserine lactone (acyl-HSL) synthase genes, lasI from Pseudomonas aeruginosa and yenI from Yersinia enterocolitica, were introduced into tobacco, individually and in combination. Liquid chromatograph-tandem mass spectrometry and thin-layer chromatography confirmed products of lasI and yenI activity in single and cotransformants. Cotransformants expressing plastid-localized LasI and YenI synthases produced the major acyl-HSLs for each synthase in all tissues tested. Total acyl-HSL signals accumulated in leaf tissue up to 3 pmol/mg of fresh weight, half as much in stem tissue, and approximately 10-fold less in root tissues. Acyl-HSLs were present in aqueous leaf washes from greenhouse-grown transgenic plants. Transgenic lines grown for 14 days under axenic conditions produced detectable levels of acyl-HSLs in root exudates. Ethyl acetate extractions of rhizosphere and nonrhizosphere soil from transgenically grown plants contained active acyl-HSLs, whereas plant-free soil or rhizosphere and nonrhizosphere soil from wild-type plants lacked detectable amounts of acyl-HSLs. This work shows that bioactive acyl-HSLs are exuded from leaves and roots and accumulate in the phytosphere of plants engineered to produce acyl-HSLs. These data further suggest that plants that are bioengineered to synthesize acyl-HSLs can foster beneficial plant-bacteria communications or deter deleterious interactions. Therefore, it is feasible to use bioengineered plants to supplement soils with specific acyl-HSLs to modulate bacterial phenotypes and plant-associated bacterial community structures.

4-Butyrolactone↗

Quorum sensing regulates dpsA and the oxidative stress response in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal human tropical disease. The non-specific DNA-binding protein DpsA plays a key role in protecting B. pseudomallei from oxidative stress mediated, for example, by organic hydroperoxides. The regulation of dpsA expression is poorly understood but one possibility is that it is regulated in a cell population density-dependent manner via N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) since a lux-box motif has been located within the dpsA promoter region. Using liquid chromatography and tandem mass spectrometry, it was first established that B. pseudomallei strain PP844 synthesizes AHLs. These were identified as N-octanoylhomoserine lactone (C8-HSL), N-(3-oxooctanoyl)homoserine lactone (3-oxo-C8-HSL), N-(3-hydroxyoctanoyl)-homoserine lactone (3-hydroxy-C8-HSL), N-decanoylhomoserine lactone (C10-HSL), N-(3-hydroxydecanoyl) homoserine lactone (3-hydroxy-C10-HSL) and N-(3-hydroxydodecanoyl)homoserine lactone (3-hydroxy-C12-HSL). Mutation of the genes encoding the LuxI homologue BpsI or the LuxR homologue BpsR resulted in the loss of C8-HSL and 3-oxo-C8-HSL synthesis, demonstrating that BpsI was responsible for directing the synthesis of these AHLs only and that bpsI expression and hence C8-HSL and 3-oxo-C8-HSL production depends on BpsR. In bpsI, bpsR and bpsIR mutants, dpsA expression was substantially down-regulated. Furthermore, dpsA expression in Escherichia coli required both BpsR and C8-HSL. bpsIR-deficient mutants exhibited hypersensitivity to the organic hydroperoxide tert-butyl hydroperoxide by displaying a reduction in cell viability which was restored by provision of exogenous C8-HSL (bpsI mutant only), by complementation with the bpsIR genes or by overexpression of dpsA. These data indicate that in B. pseudomallei, QS regulates the response to oxidative stress at least in part via the BpsR/C8-HSL-dependent regulation of DpsA.

4-Butyrolactone↗

The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa.

LecA (PA-IL) is a cytotoxic lectin and adhesin produced by Pseudomonas aeruginosa which binds hydrophobic galactosides with high specificity and affinity. By using a lecA-egfp translation fusion and immunoblot analysis of the biofilm extracellular matrix, we show that lecA is expressed in biofilm-grown cells. In static biofilm assays on both polystyrene and stainless steel, biofilm depth and surface coverage was reduced by mutation of lecA and enhanced in the LecA-overproducing strain PAO-P47. Biofilm surface coverage by the parent strain, PAO-P47 but not the lecA mutant on steel coupons was also inhibited by growth in the presence of either isopropyl-beta-D-thiogalactoside (IPTG) or p-nitrophenyl-alpha-D-galactoside (NPG). Furthermore, mature wild-type biofilms formed in the absence of these hydrophobic galactosides could be dispersed by the addition of IPTG. In contrast, addition of p-nitrophenyl-alpha-L-fucose (NPF) which has a high affinity for the P. aeruginosa LecB (PA-IIL) lectin had no effect on biofilm formation or dispersal. Planktonic growth of P. aeruginosa PAO1 was unaffected by the presence of IPTG, NPG or NPF, nor was the strain able to utilize these sugars as carbon sources, suggesting that the observed effects on biofilm formation were due to the competitive inhibition of LecA-ligand binding. Similar results were also obtained for biofilms grown under dynamic flow conditions on steel coupons, suggesting that LecA contributes to P. aeruginosa biofilm architecture under different environmental conditions.

Adhesins, Bacterial↗