High-Throughput Microfluidic Method To Study Biofilm Formation and Host-Pathogen Interactions in Pathogenic Escherichia coli

Biofilm formation and host-pathogen interactions are frequently studied using multiwell plates; however, these closed systems lack shear force, which is present at several sites in the host, such as the intestinal and urinary tracts. Recently, microfluidic systems that incorporate shear force and very small volumes have been developed to provide cell biology models that resemble in vivo conditions. Therefore, the objective of this study was to determine if the BioFlux 200 microfluidic system could be used to study host-pathogen interactions and biofilm formation by pathogenic Escherichia coli. Strains of various pathotypes were selected to establish the growth conditions for the formation of biofilms in the BioFlux 200 system on abiotic (glass) or biotic (eukaryotic-cell) surfaces. Biofilm formation on glass was observed for the majority of strains when they were grown in M9 medium at 30°C but not in RPMI medium at 37°C. In contrast, HRT-18 cell monolayers enhanced binding and, in most cases, biofilm formation by pathogenic E. coli in RPMI medium at 37°C. As a proof of principle, the biofilm-forming ability of a diffusely adherent E. coli mutant strain lacking AIDA-I, a known mediator of attachment, was assessed in our models. In contrast to the parental strain, which formed a strong biofilm, the mutant formed a thin biofilm on glass or isolated clusters on HRT-18 monolayers. In conclusion, we describe a microfluidic method for high-throughput screening that could be used to identify novel factors involved in E. coli biofilm formation and host-pathogen interactions under shear force.     

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm² to 645.0 cm²) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm². The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm²) to 1.2 to 33.6% for toilet seat surfaces (700 cm²), with detection limits of 3.5 log₁₀ and 4.0 log₁₀ RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.     

Complex Relationships amongst Parasite Load and Escape Behaviour in an Insular Lizard

Economic escape models predict escape decisions of prey which are approached by predators. Flight initiation distance (FID, predator–prey distance when prey begins to flee) and distance fled (DF) are major variables used to characterize escape responses. In optimal escape theory, FID increases as cost of not fleeing also increases. Moreover, FID decreases as cost of fleeing increases, due to lost opportunities to perform activities that may increase fitness. Finally, FID further increases as the prey's fitness increases. Some factors, including parasitism, may affect more than one of these predictors of FID. Initially, parasitized prey may have lower fitness as well as impaired locomotor ability, which would avoid predation and/or reduce their foraging ability, further decreasing the opportunity of fleeing. For example, if parasites decrease body condition, prey fitness is reduced and escape ability may be impaired. Hence, the overall influence of parasitism on FID is difficult to predict. We examined relationships between escape decisions and different traits: parasite load, body size and body condition in the Balearic lizard, Podarcis lilfordi. Lizards that showed higher haemogregarines load had longer FID and shorter DF. Although results did not confirm our initial predictions made on the basis of optimal escape theory, our findings suggest that parasites can alter several aspects of escape behaviour in a complex way.     

The lifestyle of prokaryotic organisms influences the repertoire of promiscuous enzymes

The metabolism of microbial organisms and its diversity are partly the result of an adaptation process to the characteristics of the environments that they inhabit. In this work, we analyze the influence of lifestyle on the content of promiscuous enzymes in 761 nonredundant bacterial and archaeal genomes. Promiscuous enzymes were defined as those proteins whose catalytic activities are defined by two or more different Enzyme Commission (E.C.) numbers. The genomes analyzed were categorized into four lifestyles for their exhaustive comparisons: free‐living, extremophiles, pathogens, and intracellular. From these analyses we found that free‐living organisms have larger genomes and an enrichment of promiscuous enzymes. In contrast, intracellular organisms showed smaller genomes and the lesser proportion of promiscuous enzymes. On the basis of our data, we show that the proportion of promiscuous enzymes in an organism is mainly influenced by the lifestyle, where fluctuating environments promote its emergence. Finally, we evidenced that duplication processes occur preferentially in metabolism of free‐living and extremophiles species. Proteins 2015; 83:1625–1631. © 2015 Wiley Periodicals, Inc.     

Monomeric and Dimeric 9‐O Anthraquinone and Phenanthryl Derivatives of Cinchona Alkaloids as Chiral Solvating Agents for the NMR Enantiodiscrimination of Chiral Hemiesters

Mono‐ and bis‐alkaloid chiral auxiliaries with anthraquinone or phenanthryl cores were probed as chiral solvating agents (CSAs) for the enantiodiscrimination of chiral cyclic hemiesters. The dimeric anthraquinone derivative and the monomeric phenanthryl one showed remarkable efficiency in the nuclear magnetic resonance (NMR) differentiation of enantiomeric mixtures of hemiesters. An anthraquinone analogous with a single alkaloid unit was remarkably less effective. The conformational prevalence of the chiral auxiliaries were ascertained by NMR. Chirality 27:693–699, 2015. © 2015 Wiley Periodicals, Inc.     

Cofeeding intra‐ and interspecific transmission of an emerging insect‐borne rickettsial pathogen

Cat fleas (Ctenocephalides felis) are known as the primary vector and reservoir of Rickettsia felis, the causative agent of flea‐borne spotted fever; however, field surveys regularly report molecular detection of this infectious agent from other blood‐feeding arthropods. The presence of R. felis in additional arthropods may be the result of chance consumption of an infectious bloodmeal, but isolation of viable rickettsiae circulating in the blood of suspected vertebrate reservoirs has not been demonstrated. Successful transmission of pathogens between actively blood‐feeding arthropods in the absence of a disseminated vertebrate infection has been verified, referred to as cofeeding transmission. Therefore, the principal route from systemically infected vertebrates to uninfected arthropods may not be applicable to the R. felis transmission cycle. Here, we show both intra‐ and interspecific transmission of R. felis between cofeeding arthropods on a vertebrate host. Analyses revealed that infected cat fleas transmitted R. felis to naïve cat fleas and rat fleas (Xenopsylla cheopis) via fleabite on a nonrickettsemic vertebrate host. Also, cat fleas infected by cofeeding were infectious to newly emerged uninfected cat fleas in an artificial system. Furthermore, we utilized a stochastic model to demonstrate that cofeeding is sufficient to explain the enzootic spread of R. felis amongst populations of the biological vector. Our results implicate cat fleas in the spread of R. felis amongst different vectors, and the demonstration of cofeeding transmission of R. felis through a vertebrate host represents a novel transmission paradigm for insect‐borne Rickettsia and furthers our understanding of this emerging rickettsiosis.     

Tracing the genetic origin of Europe's first farmers reveals insights into their social organization

Farming was established in Central Europe by the Linearbandkeramik culture (LBK), a well-investigated archaeological horizon, which emerged in the Carpathian Basin, in today''s Hungary. However, the genetic background of the LBK genesis is yet unclear. Here we present 9 Y chromosomal and 84 mitochondrial DNA profiles from Mesolithic, Neolithic Starčevo and LBK sites (seventh/sixth millennia BC) from the Carpathian Basin and southeastern Europe. We detect genetic continuity of both maternal and paternal elements during the initial spread of agriculture, and confirm the substantial genetic impact of early southeastern European and Carpathian Basin farming cultures on Central European populations of the sixth–fourth millennia BC. Comprehensive Y chromosomal and mitochondrial DNA population genetic analyses demonstrate a clear affinity of the early farmers to the modern Near East and Caucasus, tracing the expansion from that region through southeastern Europe and the Carpathian Basin into Central Europe. However, our results also reveal contrasting patterns for male and female genetic diversity in the European Neolithic, suggesting a system of patrilineal descent and patrilocal residential rules among the early farmers.     

The Activator of Apoptosis Smac-DIABLO Acts as a Tetramer in Solution

Smac-DIABLO in its mature form (20.8 kDa) binds to baculoviral IAP repeat (BIR) domains of inhibitor of apoptosis proteins (IAPs) releasing their inhibitory effects on caspases, thus promoting cell death. Despite its apparent molecular mass (∼100 kDa), Smac-DIABLO was held to be a dimer in solution, simultaneously targeting two distinct BIR domains. We report an extensive biophysical characterization of the protein alone and in complex with the X-linked IAP (XIAP)-BIR2-BIR3 domains. Our data show that Smac-DIABLO adopts a tetrameric assembly in solution and that the tetramer is able to bind two BIR2-BIR3 pairs of domains. Our small-angle x-ray scattering-based tetrameric model of Smac-DIABLO/BIR2-BIR3 highlights some conformational freedom of the complex that may be related to optimization of IAPs binding.