Investigation of the Iron(III)-Siderophore Binding Properties of Three Bacterial Periplasmic Binding Proteins

Bacteria possess complex machinery for uptake of essential iron. Studying iron-uptake provides insight into requirements for bacterial growth, and for development of applications, including novel antibiotics and diagnostic tools. The presented project investigates the binding of periplasmic binding proteins CeuE, FepB and VctP with a range of iron(III)-siderophore and siderophore-mimic compounds. CeuE was shown to bind iron(III)-n-LICAM (n= 6, 8) (N,N'-hexane-1,6-diylbis (2,3-dihydroxybenzamide and N,N'-octane-1,8-diylbis (2,3-dihydroxybenzamide)) tetradentate siderophore mimics, with Λ-configuration in both crystal and solution phase, with binding constants of 33 ±8 and 58 ± 8 nM respectively. Comparing these results to those for iron(III)-n-LICAM (n= 4, 5) (N,N'-butane-1,4-diylbis (2,3-dihydroxybenzamide) and N,N'-pentane-1,5-diylbis (2,3-dihydroxybenzamide)) revealed that the highest affinity was found for a five-atom linker. Mutagenesis of His 227 and Tyr 288 that coordinate the iron(III)-centre, proved that Tyr 288 is essential for iron(III)-n-LICAM binding to CeuE. Binding affinity is slightly reduced for all iron(III)-n-LICAM (n= 4, 5, 6, 8) ligands with mutation to His 227. CeuE-H227L-iron(III)-5-LICAM crystal structure determination revealed that iron(III)-5-LICAM bound in the Λ-configuration with one aqua-ligand. Salmochelin mimic siderophores Sal-n-LICAM (n= 4, 5) were synthesised and iron(III)-binding established via Job plot. For both compounds, equilibria of 1:1 and 3:2 ligand:metal ratios were observed, with 3:2 predominating over time. CeuE bound both iron(III)-complexes weakly in the Λ-configuration. Iron(III)-Sal-5-LICAM bound with higher affinity (511 ±76 nM) than iron(III)-Sal-4-LICAM (15.6 ± 2.3 µM). FepB was overexpressed, purified, and its siderophore-binding profile compared with that of CeuE and VctP. It was shown that FepB bound iron(III)-enterobactin with nanomolar affinity, and had micromolar affinity for tetradentate catecholate complexes. CeuE bound tetradentate catecholate complexes with nanomolar affinity, and iron(III)-enterobactin with micromolar affinity. VctP bound tetradentate catecholate complexes with picomolar affinity, iron(III)-enterobactin with mid-nanomolar affinity, and iron(III)-Sal-n-LICAM (n= 4, 5) with low-nanomolar affinity. All three proteins bound iron(III)-MECAM (1,3,5-N,N',N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) with low nanomolar affinity

The effects of processing and mastication on almond lipid bioaccessibility using novel methods of in vitro digestion modelling and micro-structural analysis

A number of studies have demonstrated that consuming almonds increases satiety but does not result in weight gain, despite their high energy and lipid content. To understand the mechanism of almond digestion, in the present study, we investigated the bioaccessibility of lipids from masticated almonds during in vitro simulated human digestion, and determined the associated changes in cell-wall composition and cellular microstructure. The influence of processing on lipid release was assessed by using natural raw almonds (NA) and roasted almonds (RA). Masticated samples from four healthy adults (two females, two males) were exposed to a dynamic gastric model of digestion followed by simulated duodenal digestion. Between 7·8 and 11·1 % of the total lipid was released as a result of mastication, with no significant differences between the NA and RA samples. Significant digestion occurred during the in vitro gastric phase (16·4 and 15·9 %) and the in vitro duodenal phase (32·2 and 32·7 %) for the NA and RA samples, respectively. Roasting produced a smaller average particle size distribution post-mastication; however, this was not significant in terms of lipid release. Light microscopy showed major changes that occurred in the distribution of lipid in all cells after the roasting process. Further changes were observed in the surface cells of almond fragments and in fractured cells after exposure to the duodenal environment. Almond cell walls prevented lipid release from intact cells, providing a mechanism for incomplete nutrient absorption in the gut. The composition of almond cell walls was not affected by processing or simulated digestion

Impact of cell wall encapsulation of almonds on in vitro duodenal lipolysis

Although almonds have a high lipid content, their consumption is associated with reduced risk of cardiovascular disease. One explanation for this paradox could be limited bioaccessibility of almond lipids due to the cell wall matrix acting as a physical barrier to digestion in the upper gastrointestinal tract. We aimed to measure the rate and extent of lipolysis in an in vitro duodenum digestion model, using raw and roasted almond materials with potentially different degrees of bioaccessibility. The results revealed that a decrease in particle size led to an increased rate and extent of lipolysis. Particle size had a crucial impact on lipid bioaccessibility, since it is an indicator of the proportion of ruptured cells in the almond tissue. Separated almond cells with intact cell walls showed the lowest levels of digestibility. This study underlines the importance of the cell wall for modulating lipid uptake and hence the positive health benefits underlying almond consumption

Organizational Communication and Individual Behavior: Implications for Supply Chain Risk Management

Risk is a significant issue for supply chain managers. Not only must they contend with multiple dimensions of risk in decision‐making, they must reconcile decision‐making with broader organizational interests. This study examines the influence of organizational communication regarding supply chain risk on individual decision‐making strategies and the perceptions of risk. A multi‐stage experimental design is applied, in which decision‐makers make decisions across three dimensions of risk and adjust their risk‐taking behavior after being presented with organizational communication regarding supply chain risk levels. The relationship between organizational communication and the perceptions of supply chain risk is then explored after decision‐makers are allowed to adjust their supply chain strategies. The results suggest that decision‐makers adapt sourcing strategies in response to organizational communication regarding supply chain risk. Specifically, they make riskier decisions when the organization communicates improvements in supply chain risk levels. However, when given specific instructions to reduce risk, they do not adjust their supply chain strategies

A Salmochelin S4-inspired Ciprofloxacin Trojan Horse Conjugate

A novel ciprofloxacin-siderophore Trojan Horse antimicrobial was prepared by incorporating key design features of salmochelin S4, a stealth siderophore that evades mammalian siderocalin capture via its glycosylated catechol units. As-sessment of the antimicrobial activity of the conjugate revealed that attachment of the salmochelin mimic resulted in decreased potency, compared to ciprofloxacin, against two Escherichia coli strains, K12 and Nissle 1917, in both iron-replete and deplete conditions. This observation could be attributed to a combination of reduced DNA gyrase inhibi-tion, as confirmed by in vitro DNA gyrase assays, and reduced bacterial uptake. Uptake was monitored using radio-labelling with iron-mimetic 67Ga3+, which revealed limited cellular uptake in E. coli K12. In contrast, previously reported staphyloferrin-based conjugates displayed measurable uptake in analogous 67Ga3+, labelling studies. These results suggest that in designing Trojan Horse antimicrobials, the choice of siderophore and the nature and length of the link-er remains a significant challenge

Mimicking salmochelin S1 and the interactions of its Fe(III) complex with periplasmic iron siderophore binding proteins CeuE and VctP

A mimic of the tetradentate stealth siderophore salmochelin S1, was synthesised, characterised and shown to form Fe(III) complexes with ligand-to-metal ratios of 1:1 and 3:2. Circular dichroism spectroscopy confirmed that the periplasmic binding proteins CeuE and VctP of Campylobacter jejuni and Vibrio cholerae, respectively, bind the Fe(III) complex of the salmochelin mimic by preferentially selecting Λ-configured Fe(III) complexes. Intrinsic fluorescence quenching studies revealed that VctP binds Fe(III) complexes of the mimic and structurally-related catecholate ligands, such as enterobactin, bis(2, 3-dihydroxybenzoyl-l-serine) and bis(2, 3-dihydroxybenzoyl)-1, 5-pentanediamine with higher affinity than does CeuE. Both CeuE and VctP display a clear preference for the tetradentate bis(catecholates) over the tris(catecholate) siderophore enterobactin. These findings are consistent with reports that V. cholerae and C. jejuni utilise the enterobactin hydrolysis product bis(2, 3-dihydroxybenzoyl)-O-seryl serine for the acquisition of Fe(III) and suggest that the role of salmochelin S1 in the iron uptake of enteric pathogens merits further investigation

Mongooses (\u3ci\u3eUrva auropunctata\u3c/i\u3e) as reservoir hosts of leptospira species in the United States Virgin Islands, 2019–2020

During 2019–2020, the Virgin Islands Department of Health investigated potential animal reservoirs of Leptospira spp., the bacteria that cause leptospirosis. In this cross-sectional study, we investigated Leptospira spp. exposure and carriage in the small Indian mongoose (Urva auropunctata, syn: Herpestes auropunctatus), an invasive animal species. This study was conducted across the three main islands of the U.S. Virgin Islands (USVI), which are St. Croix, St. Thomas, and St. John. We used the microscopic agglutination test (MAT), fluorescent antibody test (FAT), real-time polymerase chain reaction (lipl32 rt-PCR), and bacterial culture to evaluate serum and kidney specimens and compared the sensitivity, specificity, positive predictive value, and negative predictive value of these laboratory meth-ods. Mongooses (n = 274) were live-trapped at 31 field sites in ten regions across USVI and humanely euthanized for Leptospira spp. testing. Bacterial isolates were sequenced and evaluated for species and phylogenetic analysis using the ppk gene. Anti-Leptospira spp. antibodies were detected in 34% (87/256) of mongooses. Reactions were observed with the following serogroups: Sejroe, Icterohaemorrhagiae, Pyrogenes, Mini, Cynopteri, Australis, Hebdomadis, Autumnalis, Mankarso, Pomona, and Ballum. Of the kidney specimens exam-ined, 5.8% (16/270) were FAT-positive, 10% (27/274) were culture-positive, and 12.4% (34/ 274) were positive by rt-PCR. Of the Leptospira spp. isolated from mongooses, 25 were L. borgpetersenii, one was L. interrogans, and one was L. kirschneri. Positive predictive values of FAT and rt-PCR testing for predicting successful isolation of Leptospira by culture were 88% and 65%, respectively. The isolation and identification of Leptospira spp. in mongooses highlights the potential role of mongooses as a wildlife reservoir of leptospirosis; mongooses could be a source of Leptospira spp. infections for other wildlife, domestic animals, and humans

Citizen Journalism at the Margins

Amidst burgeoning literature on citizen journalism, we still know relatively little about how and why genuinely marginalised groups seek to use this form of reporting to challenge their exclusion. In this article, we aim to address this gap by analysing two UK citizen journalism initiatives emanating from The Big Issue Foundation, a national homeless organisation, and Access Dorset, a regional charity for disabled and elderly people. These case studies are united by the authors’ involvement in both instances, primarily through designing and delivering bespoke citizen journalism education and mentoring. Based on over 40 hours of interviews with participants of the workshops and 36 hours of participant observation, we analyse the challenges participants faced in their journey to become citizen journalists. This included: low self-esteem, physical health and mental wellbeing, the need for accessible and adaptable technology, and overcoming fear associated with assuming a public voice. We also analyse marginalised groups’ attitudes to professional journalism and education, and its role in shaping journalistic identity and self-empowerment. Whilst demonstrably empowering and esteem building,our participants were acutely aware of societal power relations that were seemingly still beyond their ability to influence. Those who are marginalised are, nevertheless, in the best position to use citizen journalism as a conduit for social change, we argue - though challenges remain even at the grassroots level to foster and sustain participatory practices

Frustration Tolerance and Personality Traits in Patients With Substance Use Disorders

Previous research has suggested the prevalence of certain personality traits, some of which are related to a disorganized attachment, in substance abuse disorders. Further, frustration tolerance (FT) has been proposed as an important factor in addiction, both at the inception—following the “self-medication” hypothesis—and regarding treatment compliance. In turn, an inadequate response to frustrating events has been also associated with a disrupted attachment. Our goal is to explore the mediational role of FT in the relationship between personality traits and two different treatments for substance addiction: therapeutic community (TC) and ambulatory treatment (AT). Eighty-four subjects with substance abuse disorder were recruited in total (22 female), including 46 volunteers (13 female) in TC and 38 (9 female) in AT. They were assessed with Rosenzweig’s test for FT and the Millon Clinical Multiaxial Inventory-III (MCMI-III) test to evaluate personality factors. By comparing with a control sample (335 volunteers, 268 female), we found that FT was lower in patients. Between therapeutic groups, FT was significantly lower in TC. Depressive, antisocial, sadistic, negativistic, schizotypal, borderline, paranoid, anxiety, dysthymia, alcohol use, drug use, posttraumatic stress disorder (PTSD), thought disorder, and delusional disorder traits were suggestive of pathology in the clinical samples and were significantly different between control, AT, and TC groups. Further, anxiety and PTSD traits were higher in TC than in AT. A mediational analysis revealed that the effect of anxiety and PTSD scales on therapeutic group was partially mediated by FT. In conclusion, FT and its interplay with personality traits commonly related to disorganized attachment (anxiety and PTSD) might be important factors to consider within therapeutic programs for persons with substance addiction.We also acknowledge the collaboration of the users and personnel of Fundación Proyecto Hombre Navarra and Asociación Proyecto Hombre Granada for making possible this research. This project was funded by Fundación Caja de Ahorros de Navarra (grant number 70721) and the Spanish Ministerio de Sanidad, Servicios Sociales e Igualdad (2016/057)