Mutational Analysis of the Quorum-Sensing Receptor LasR Reveals Interactions that Govern Activation and Inhibition by Nonlactone Ligands

SummaryGram-negative bacteria use N-acyl L-homoserine lactone (AHL) quorum-sensing (QS) signals to regulate the expression of myriad phenotypes. Non-native AHL analogs can strongly attenuate QS receptor activity and thereby QS signaling; however, we currently lack a molecular understanding of the mechanisms by which most of these compounds elicit their agonistic or antagonistic profiles. In this study, we investigated the origins of striking activity profile switches (i.e., receptor activator to inhibitor, and vice versa) observed upon alteration of the lactone head group in certain AHL analogs. Reporter gene assays of mutant versions of the Pseudomonas aeruginosa QS receptor LasR revealed that interactions between the ligands and Trp60, Tyr56, and Ser129 govern whether these ligands behave as LasR activators or inhibitors. Using this knowledge, we propose a model for the modulation of LasR by AHL analogs—encompassing a subtly different interaction with the binding pocket to a global change in LasR conformation

Survey on the prevalence of dyspepsia and practices of dyspepsia management in rural Eastern Uganda

Aim: To investigate the current prevalence and management of dyspepsia in rural Eastern Uganda. Methods: Residents older than 18 years of age across 95 study sites in Namutumba District, Eastern Uganda were surveyed. Each respondent was administered a questionnaire about dyspepsia and pertinent health-seeking behaviors. Health workers at 12 different clinics were also assessed on their competence in managing dyspepsia. Proportion-based analysis was used to determine self-reported outcome variables reported in this study, including: prevalence of dyspepsia; breakdown of symptoms; initial diagnosis location; management strategies; and appropriate medication usage. Results: 397 residents (average age of 41.2 years) participated in this study (54.4% males, 45.6% females). 57.9% self-reported currently having dyspepsia, of average duration 4.5 years. Of this subset, 87% reported experiencing epigastric pain, and 42.2% believed that ulcers were wounds in the stomach. Only 3% of respondents had heard of Conclusion: There is a high incidence of dyspepsia in Eastern Uganda, and current management strategies are poor and inconsistent, and may contribute to antibiotic resistance. Further studies are needed to investigate the causes of dyspepsia to guide appropriate management

In search of heterotopia? Visitor motivations to an English Cathedral

According to Shackley (2002, after Foucault, 1986), the motivation of most visitors to cathedral is the search for heterotopia, which is defined as a sense of timelessness and spirituality. Shackley goes on to argue that this search for heterotopia is a conscious, even if sometimes not articulated, attempt by visitors to connect to an unchanging and transcendental space that provides spiritual meaning in a life of transient and ephemeral values. Shackley (2002:350) proposes that visitors to cathedrals recognise them as sacred space and are influenced by them, even if they cannot articulate or recognise that effect. This paper explores the idea that many visitors to sacred sites and cathedrals in particular, are motivated to do so by a search of spirituality, but that this search is acting as a subconscious, unspoken motivation, which may be hidden by more rational motivations such as curiosity, a desire to learn, or an interest in history and architecture. This paper argues that although more than a third of visitors to Chichester Cathedral are motivated by a search for spirituality or heterotopia as described by Shackley (2002), their motivation is often not the result of a conscious decision, but instead appears to be the result of a subconscious motivation. This finding also suggests that a significant proportion of the tourists that, according to Sharpley and Sundaran (2005), would claim to be motivated by curiosity or a desire to learn when visiting sacred or pilgrimage sites (the ‘Tourist trail followers’ and the ‘Practitioners’), may actually be subconsciously seeking some sense of spiritual experience or fulfilment. This proposition is based on a comparison between what visitors to Chichester Cathedral articulated as their primary motivation to visit the site, and those aspects that they found more satisfying from their visit, as well as the adjectives used to describe the Cathedral after their visit. This theory of subconscious motivation is developed from research on place meaning by Young (1999) and on consumer narratives of cathedral visitors by Voase (2007), and suggests that place meanings and personal narratives for cathedrals are socially constructed and attributed to the place according to the visitor’s motivations. In this case, place meaning is acting as a mechanism to create intrinsic benefits that satisfy the visitor’s motivational needs, with this process operating at an individual, subconscious level (Young, 1999; Pearce and Caltabiano, 1983). Thus, this paper concludes that a significant proportion of the non-religious visitors to cathedrals who argue that their motivations do not include the expectation of finding a sense of spirituality are justifying the benefits derived from visiting these sites by creating place meanings that have a spiritual dimension attached to it. This spiritual dimension arises from the subconscious acceptance of, and the associated behaviour connected to, the sanctity of space that visitors attach to the place

255 In vivo detection of non-occlusive thrombi in drug-eluting stents by scintigraphy and radio-labelled annexin V in a rabbit model

IntroductionThrombi in contact with non re-endothelialized stent struts associated with drug-eluting stent (DES) thrombosis. Hence, detection of thrombi in DES could help to evaluate the risk of DES thrombosis. Annexin V radio-labelled with 99mTechnetium (99mTc) is a radio-tracer with a high affinity for activated platelets.ObjectivesOur objectives were: 1) to develop an animal model of non-occlusive thrombosis of stents, 2) to evaluate the ability of annexin V 99mTc for the detection of in-stent thrombi using scintigraphy.MethodsRight carotid arteries of NZW rabbits (n=14) fed a high cholesterol diet were implanted with overlapping DES (n=7) or bare-metal stents (BMS; n=7). Four weeks after stent implantation, rabbits underwent a first scintigraphy 3 hours after injection of 200 MBq of radio-labelled annexin V 99mTc. At the end of the first scintigraphy, a suture was placed surgically proximal to the stented carotid arteries in order to induce a thrombus-prone flow limiting stenosis. Four days later, a second scintigraphy was performed. After the second scintigraphy, stents were excised, imaged ex vivo and then fixed for histological examination and scanning electron microscopy (SEM).ResultsActivities measured in vivo in the stented carotid arteries after injection of annexin V 99mTc were higher on the second scintigraphy after creation of a surgical stenosis as compared to the first scintigraphy (0.24 vs. 0.15 counts/pixel/MBq, respectively; p<0.05). On the second scintigraphy, activities were higher in DES vs. BMS (0.26 vs. 0.19 counts/pixel/MBq, respectively; p < 0.005). High activities measured in stents in vivo were associated with the detection of thrombi on corresponding histological sections and SEM.ConclusionsIn this work, we developed a rabbit model of non-occlusive thrombosis of stents in carotid arteries. In this model, in-stent thrombi could be detected using annexin V 99mTc scintigraphy

Peristrut microhemorrhages: a possible cause of in-stent neoatherosclerosis?

AbstractBackgroundIn-stent neoatherosclerosis is characterized by the delayed appearance of markers of atheroma in the subintima, but the pathophysiology underlying this new disease entity remains unclear.Methods and resultsWe collected 20 human coronary artery stents by removal from explanted hearts. The mean duration of stent implantation was 34 months. In all samples, neoatherosclerosis was detected, particularly in peristrut areas. It consisted of foam cells and cholesterol clefts, with or without calcification, associated with neovascularization. Iron and glycophorin-A were present in peristrut areas, as well as autofluorescent ceroids. Moreover, in response to neoatherosclerosis, tertiary lymphoid organs (tissue lymphoid clusters) often developed in the adventitia. Some of these features could be reproduced in an experimental carotid stenting model in rabbits fed a high-cholesterol diet. Foam cells were present in all samples, and peristrut red blood cells (RBCs) were also detected, as shown by iron deposits and Bandeiraea simplicifiola isolectin-B4 staining of RBC membranes. Finally, in silico models were used to evaluate the compliance mismatch between the rigid struts and the distensible arterial wall using finite element analysis. They show that stenting approximately doubles the local von Mises stress in the intimal layer.ConclusionsWe show here that stent implantation both in human and in rabbit arteries is characterized by local peristrut microhemorrhages and finally by both cholesterol accumulation and oxidation, triggering together in-stent neoatherosclerosis. Our data indicate that these processes are likely initiated by an increased mechanical stress due to the compliance mismatch between the rigid stent and the soft wall

Relationships among rat numbers, abundance of oil palm fruit and damage levels to fruit in an oil palm plantation.

The relationships between vertebrate pests and crop damage are often complex and difficult to study. In palm oil plantations rodents remain the major pests, causing substantial monetary losses. The present study examined the numerical and functional responses of rodents to changes in the availability of oil palm fruit and the damage associated with that response. For the study, 200 traps were set in pairs on a 10 × 10 trapping grid for 3 consecutive nights in each of 6 study plots at 8-week intervals in a 2569 ha oil palm plantation at Labu, Negeri Sembilan state in Peninsular Malaysia over 14 months. A total of 1292 individual rats were captured over 25 200 trap-nights. Animals were identified, aged, sexed, weighed and measured. An index of the relative abundance of rats was calculated based on trapping success. Damage to infructescences was assessed at each trap point. Regardless of the age of palms, there were positive and significant relationships between the relative abundance of rats and numbers of infructescences. The levels of damage to infructescences were significantly correlated with the relative abundance of rats. A steep increase in damage was observed with an increase in mature infructescences, indicating a feeding preference of rats for mature infructescences. For both males and females of all rat species, there were weak and non-significant correlations between body condition and infructescence numbers. These results indicated that there was a numerical and a functional response by rats to the availability of palm fruit and a resulting increase in depredation of oil palm fruits. The ways in which this information might aid in future pest control are discussed

A2 gene of Old World cutaneous Leishmania is a single highly conserved functional gene

BACKGROUND: Leishmaniases are among the most proteiform parasitic infections in humans ranging from unapparent to cutaneous, mucocutaneous or visceral diseases. The various clinical issues depend on complex and still poorly understood mechanisms where both host and parasite factors are interacting. Among the candidate factors of parasite virulence are the A2 genes, a family of multiple genes that are developmentally expressed in species of the Leishmania donovani group responsible for visceral diseases (VL). By contrast, in L. major determining cutaneous infections (CL) we showed that A2 genes are present in a truncated form only. Furthermore, the A2 genomic sequences of L. major were considered subsequently to represent non-expressed pseudogenes [1]. Consequently, it was suggested that the structural and functional properties of A2 genes could play a role in the differential tropism of CL and VL leishmanias. On this basis, it was of importance to determine whether the observed structural/functional particularities of the L. major A2 genes were shared by other CL Leishmania, therefore representing a proper characteristic of CL A2 genes as opposed to those of VL isolates. METHODS: In the present study we amplified by PCR and sequenced the A2 genes from genomic DNA and from clonal libraries of the four Old World CL species comparatively to a clonal population of L. infantum VL parasites. Using RT-PCR we also amplified and sequenced A2 mRNA transcripts from L. major. RESULTS: A unique A2 sequence was identified in Old World cutaneous Leishmania by sequencing. The shared sequence was highly conserved among the various CL strains and species analysed, showing a single polymorphism C/G at position 58. The CL A2 gene was found to be functionally transcribed at both parasite stages. CONCLUSION: The present study shows that cutaneous strains of leishmania share a conserved functional A2 gene. As opposed to the multiple A2 genes described in VL isolates, the CL A2 gene is unique, lacking most of the nucleotide repeats that constitute the variable region at the 5'end of the VL A2 sequences. As the variable region of the VL A2 gene has been shown to correspond to a portion of the protein which is highly immunogenic, the present results support the hypothesis of a possible role of the A2 gene in the differential tropism of CL and VL leishmania parasites

Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression

Gene promoters are enriched in guanine clusters that potentially fold into quadruplex structures. Such quadruplexes were implicated in the regulation of gene expression, plausibly by interacting with transcription factors. We showed previously that homodimers of the myogenic transcription factor MyoD bound in vitro most tightly bimolecular quadruplexes of promoter sequences of muscle-specific genes. By contrast, MyoD-E47 heterodimers formed tighter complexes with d(CANNTG) E-box motifs that govern muscle gene expression. Here, we show that DNA quadruplexes enhance in vivo MyoD and E-box-driven expression of a firefly luciferase (FL) reporter gene. HEK293 cells were transfected with FL expressing p4RTK-FL vector alone or together with MyoD expressing pEMSV-MyoD plasmid, with quadruplexes of α7 integrin or sarcomeric mitochondrial creatine kinase (sMtCK) muscle gene promoters or with a combination thereof. Whereas MyoD elevated by ∼10-fold the levels of FL mRNA and protein, the DNA quadruplexes by themselves did not affect FL expression. However, together with MyoD, quadruplex DNA increased by ∼35-fold the amounts of FL mRNA and protein. Without affecting its expression, DNA quadruplexes bound MyoD in the cells. Based on these results, we propose models for the regulation of muscle gene transcription by direct interaction of MyoD with promoter quadruplex structures