Functional Amyloids Keep Quorum-sensing Molecules in Check

The mechanism by which extracellular metabolites, including redox mediators and quorum-sensing signaling molecules, traffic through the extracellular matrix of biofilms is poorly explored. We hypothesize that functional amyloids, abundant in natural biofilms and possessing hydrophobic domains, retain these metabolites. Using surface plasmon resonance, we demonstrate that the quorum-sensing (QS) molecules, 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone, and the redox mediator pyocyanin bind with transient affinity to functional amyloids from Pseudomonas (Fap). Their high hydrophobicity predisposes them to signal-amyloid interactions, but specific interactions also play a role. Transient interactions allow for rapid association and dissociation kinetics, which make the QS molecules bioavailable and at the same time secure within the extracellular matrix as a consequence of serial bindings. Retention of the QS molecules was confirmed using Pseudomonas aeruginosa PAO1-based 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone reporter assays, showing that Fap fibrils pretreated with the QS molecules activate the reporters even after sequential washes. Pyocyanin retention was validated by electrochemical analysis of pyocyanin-pretreated Fap fibrils subjected to the same washing process. Results suggest that QS molecule-amyloid interactions are probably important in the turbulent environments commonly encountered in natural habitats.     

Genetic Variants in FBN-1 and Risk for Thoracic Aortic Aneurysm and Dissection

Objectives

A recent genome wide association study (GWAS) by LeMaire et al. found that two single nucleotide polymorphisms (SNPs), rs2118181 and rs10519177 in the FBN-1 gene (encoding Fibrillin-1), were associated with thoracic aortic dissection (TAD), non-dissecting thoracic aortic aneurysm (TAA), and thoracic aortic aneurysm or dissection (TAAD); the largest effect was observed for the association of rs2118181 with TAD. We investigated whether rs2118181 and rs10519177 were associated with TAD, TAA, and TAAD in the Yale study.

Methods

The genotypes of rs2118181 and rs10519177 were determined for participants in the Yale study: 637 TAAD cases (140 TAD, 497 TAA) and 275 controls from the United States, Hungary, and Greece. The association of the genotypes with TAD, TAA and TAAD were assessed using logistic regression models adjusted for sex, age, study center and hypertension.

Results and Conclusions

In the Yale study, rs2118181 was associated with TAD: compared with non-carriers, carriers of the risk allele had an unadjusted odds ratio for TAD of 1.80 (95% CI 1.15–2.80) and they had odds ratio for TAD of 1.87 (95% CI 1.09–3.20) after adjusting for sex, age, study center and hypertension. We did not find significant differences in aortic size, a potential confounder for TAD, between rs2118181 risk variant carriers and non-carriers: mean aortic size was 5.56 (95% CI: 5.37–5.73) for risk variant carriers (CC+CT) and was 5.48 (95% CI: 5.36–5.61) for noncarriers (TT) (p = 0.56). rs2118181 was not associated with TAA or TAAD. rs10519177 was not associated with TAD, TAA, or TAAD in the Yale study. Thus, the Yale study provided further support for the association of the FBN-1 rs2118181SNP with TAD.     

Solution structure of ZASP PDZ domain; implications for sarcomere ultrastructure and enigma family redundancy

Z band alternately spliced PDZ-containing protein (ZASP) is a sarcomere Z disk protein expressed in human cardiac and skeletal muscle that is thought to be involved in a dominant familial dilated cardiomyopathy. The N-terminal PDZ domain of ZASP interacts with the C terminus of alpha-actinin-2, the major component of the Z disk, probably by forming a ternary complex with titin Z repeats. We have determined the structure of ZASP PDZ by NMR and showed that it is a classical class 1 PDZ domain that recognizes the carboxy-terminal sequence of an alpha-actinin-2 calmodulin-like domain with micromolar affinity. We also characterized the role of each component in the ternary complex ZASP/alpha-actinin-2/titin, showing that the alpha-actinin-2/ZASP PDZ interaction involves a binding surface distinct from that recognized by the titin Z repeats. ZASP PDZ structure was used to model other members of the enigma family by homology and to predict their abilities to bind alpha-actinin-2.     

Loss of Parafollicular Cells during Gravitational Changes (Microgravity,Hypergravity) and the Secret Effect of Pleiotrophin

It is generally known that bone loss is one of the most important complications for astronauts who are exposed to long-term microgravity in space. Changes in blood flow, systemic hormones, and locally produced factors were indicated as important elements contributing to the response of osteoblastic cells to loading, but research in this field still has many questions. Here, the possible biological involvement of thyroid C cells is being investigated. The paper is a comparison between a case of a wild type single mouse and a over-expressing pleiotrophin single mouse exposed to hypogravity conditions during the first animal experiment of long stay in International Space Station (91 days) and three similar mice exposed to hypergravity (2Gs) conditions. We provide evidence that both microgravity and hypergravity induce similar loss of C cells with reduction of calcitonin production. Pleiotrophin over-expression result in some protection against negative effects of gravity change. Potential implication of the gravity mechanic forces in the regulation of bone homeostasis via thyroid equilibrium is discussed.     

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis.In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10(-7)M and 10(-8)M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains.In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. -90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules.Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.     

Thermodynamic and spectroscopic investigation on the role of Met residues in Cu(II) binding to the non-octarepeat site of the human prion protein

Among the common features shared by neurodegenerative diseases there is the central role played by specific proteins or peptides which accumulate in neurons as insoluble plaques or tangles, containing abnormal amounts of redox-active metal ions, like copper and iron. In the case of transmissible spongiform encephalopathies (TSE), the involved protein is known as "prion protein" (PrP(C)) since "prions" (proteinaceous and infectious) are the agents which make TSE transmissible. It is widely accepted that PrP(C), in its wild-type form, can bind up to six Cu(II) ions, four of them in the so-called "octarepeat domain" and the others in the "fifth (non-octarepeat) binding-site". The latter domain contains two His residues, acting as anchoring sites for Cu(II) ions, and other potential binding residues, such as Lys and Met. While it is widely accepted that Lys residues do not take part in complex-formation, the role of methionines is still debated. In order to shed light on this issue, some peptides have been synthesized, either directly mimicking the sequence of the second half of the fifth binding site of human-PrP(C) (apo-form) or analogues where Met residues have been substituted by n-leucine. In addition, a series of short peptides, containing both His and Met residues in different relative positions, have been investigated, for the sake of comparison. Spectroscopic results, including NMR spectra of systems containing Ni(II) as a probe for the paramagnetic Cu(II) ion, agree on the exclusion of any direct interaction between the sulphur atom of Met residues and the Cu(II) ion already bound to His-imidazole side-chains. However, thermodynamic data show that Met-109 somewhat contributes to stability of complex species and this can be attributed to different electronic and steric effects.     

Evidence of lethal and sublethal injury in food-borne bacterial pathogens exposed to high-intensity pulsed-plasma gas discharges

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to pulsed-plasma gas discharges (PPGD). METHODS AND RESULTS: The fluorescent redox probe 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used for enumerating actively respiring cells of Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella enterica serovar Typhimurium that were suspended in sterile water at 4 degrees C and exposed to separate PPGD and heat treatments. While there was good agreement between use of respiratory staining (RS) and direct-selective agar plate counting (PC) for enumerating untreated bacteria, there were c. 1 and 3 log-unit differences in surviving cell numbers per millilitre for test organisms subjected to PPGD and heat treatments respectively, when enumerated by these different viability indicators. PPGD-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: Use of this RS method revealed that substantial subpopulations of test bacteria rendered incapable of forming colonies by separate PPGD and heat treatments may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of this RS method offers interesting perspectives on assessing established and novel microbial inactivation methods, and may also provide a better understanding of mechanisms involved in microbial inactivation induced by high-intensity PPGD treatments.     

UV reflecting vole scent marks attract a passerine, the great grey shrike Lanius excubitor

Diurnal raptors have been shown to use ultraviolet vision and UV-reflecting vole scent marks to find suitable hunting areas. We studied if a passerine species, the great grey shrike Lanius excubitor that uses voles as its primary food, may also detect prey-patches in the same way. We conducted a laboratory experiment with ten shrikes. Each individual shrike had four options to choose from: (1) scent marks with UV light, (2) scent marks without UV light, (3) clean arena with UV light, and (4) clean arena without UV light. The birds preferred the scent-marked arena with UV light as measured by the number of scans and the time spent above it. Therefore, we suggest that great grey shrike probably uses UV cues to gain information on vole locations and abundance.