Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.     

No pre-existing biases for heterospecific call traits in the frog Physalaemus enesefae

Previous phonotaxis studies with two species of the Physalaemuspustulosus species group indicated that female preferences for several heterospecific call traits resulted from sensory biases inherited from a common ancestor. In phonotaxis experiments, we determined whether Physalaemus enesefae, a distant relative of the P. pustulosus group, showed similar preferences for call traits not present in conspecific males. We presented females with a choice between the typical conspecific advertisement call and the same call to which we digitally appended a chuck from P. pustulosus, a squawk from P. freibergi, and an amplitude-modulated prefix from P. pustulatus. In addition we presented the advertisement call in doublets, a trait peculiar to P. coloradorum. We also analysed male vocal behaviour evoked in response to the same suite of stimuli. Physalaemus enesefae females did not prefer the calls of their own males with appended heterospecific traits over unmodified calls, nor conspecific calls in doublets over single calls. The lack of preference among females was not the result of a behavioural polymorphism. Female responses to repeated presentations of the same stimulus pair were not consistent. Males also did not show an enhanced vocal response to altered calls relative to the typical conspecific call. Consequently, there are no pre-existing biases for these heterospecific call traits in P. enesefae; pre-existing preferences in the P. pustulosus group could have been inherited from an ancestor not shared with P. enesefae, but data from other closely related species are needed to confirm this conclusion. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

hapConstructor: automatic construction and testing of haplotypes in a Monte Carlo framework

SUMMARY: Haplotypes carry important information that can direct investigators towards underlying susceptibility variants, and hence multiple tagging single nucleotide polymorphisms (tSNPs) are usually studied in candidate gene association studies. However, it is often unknown which SNPs should be included in haplotype analyses, or which tests should be performed for maximum power. We have developed a program, hapConstructor, which automatically builds multi-locus SNP sets to test for association in a case-control framework. The multi-SNP sets considered need not be contiguous; they are built based on significance. An important feature is that the missing data imputation is carried out based on the full data, for maximal information and consistency. HapConstructor is implemented in a Monte Carlo framework and naturally extends to allow for significance testing and false discovery rates that account for the construction process and to related individuals. HapConstructor is a useful tool for exploring multi-locus associations in candidate genes and regions. AVAILABILITY: http://www-genepi.med.utah.edu/Genie.     

Secondary reduction of preference for the sword ornament in the pygmy swordtail Xiphophorus nigrensis (Pisces: Poeciliidae)

Female mating preferences can be secondarily lost for a number of reasons. We examined the preference of female pygmy swordtails, Xiphophorus nigrensis, for the sword, a conspicuous extension of the caudal fin in some males. Females failed to show a preference for conspecific males with swords when presented with live males naturally varying in sword length, with live males of manipulated sword length, and with synthetic animations of males expressing natural variation in sword length. Females showed a significant bias against swords when presented with synthetic animations bearing supernormal sword characteristics. The reduced preference for swords, relative to closely related fish, may result from an increase in the cost of choice due to predation risk, selection against mating with heterospecifics, or changes in the spatial and contrast properties of the conspecific signal.     

Snakes and ladders: legal coercion,housing precarity,and home-making aspirations in southern England

The potential for eviction is an ordinary condition of domestic life for many in Europe and North America. This poses a challenge to anthropological theories of the state's presence in ordinary homes, which have accounted for public housing and mass displacement, but not liberalized settings where the state has ostensibly withdrawn from the home. Studies of housing precarity identify state policy and capitalist transformation among its sources, but the consequences of housing precarity for domesticity itself have not been fully explored. Private renters on a housing estate in southern England responded to the bleak prospect of eviction with home-making pursuits that would instil a sense of optimism in their homes, including mortgage-based ownership and immersive home entertainment technology. By examining the interplay between fears of eviction and home-making aspirations, this article argues that the British state's organization of legitimate coercion has a subtle but significant influence on tenants’ ethical visions of what constitutes a good home.     

Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and noninvasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP was reduced 3 to 11 times slower (depending on the tissue) than Tempol in vivo and that maximum tissue concentration varies substantially between tissues (0.6-7.2mM). For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance spectroscopy, we showed that the nitroxide reduction rate depends only weakly on cellular pO(2) in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO(2) is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo.     

Identification of novel ryanodine receptor 1 (RyR1) protein interaction with calcium homeostasis endoplasmic reticulum protein (CHERP)

The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca(2+) release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1-4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca(2+) release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.