Alleviation of restriction by DNA condensation and non-specific DNA binding ligands

During conditions of cell stress, the type I restriction and modification enzymes of bacteria show reduced, but not zero, levels of restriction of unmethylated foreign DNA. In such conditions, chemically identical unmethylated recognition sequences also occur on the chromosome of the host but restriction alleviation prevents the enzymes from destroying the host DNA. How is this distinction between chemically identical DNA molecules achieved? For some, but not all, type I restriction enzymes, alleviation is partially due to proteolytic degradation of a subunit of the enzyme. We identify that the additional alleviation factor is attributable to the structural difference between foreign DNA entering the cell as a random coil and host DNA, which exists in a condensed nucleoid structure coated with many non-specific ligands. The type I restriction enzyme is able to destroy the ‘naked’ DNA using a complex reaction linked to DNA translocation, but this essential translocation process is inhibited by DNA condensation and the presence of non-specific ligands bound along the DNA

Spaces for play:Listening to children's voices

This paper explores young children’s voices about their play spaces in one Scottish primary school. 45 children (ages 5-7 years) participated, choosing from a range of creative methods (e.g., InPhoTours, drawing, mapping) to share their voices. Using a ‘Playful Research Ethics Framework’, a developmentally appropriate framework which involved the use of visual aids, puppets, songs, Makaton symbols and discussions as well as attention to any cues of disengagement, this research aimed at achieving children’s ongoing informed assent. Four themes were identified: (a) the ‘whole child’ in the space, (b) space and relationships, (c) function of space, and (d) impact (or lack) of children’s voices about space. Children expressed differences in ownership, creativity and imagination in indoor and outdoor spaces. Further, despite perceiving there to be a lack of agency, children were willing to share their voices. There are implications for both practice and research in terms of adults willing to effectively listen to children’s voices and acting on them. This study makes original and significant contributions which have the potential to impact research and practice with young children internationally

StpA protein from Escherichia coli condenses supercoiled DNA in preference to linear DNA and protects it from digestion by DNase I and EcoKI

The nucleoid-associated protein, StpA, of Escherichia coli binds non-specifically to double-stranded DNA (dsDNA) and apparently forms bridges between adjacent segments of the DNA. Such a coating of protein on the DNA would be expected to hinder the action of nucleases. We demonstrate that StpA binding hinders dsDNA cleavage by both the non-specific endonuclease, DNase I, and by the site-specific type I restriction endonuclease, EcoKI. It requires approximately one StpA molecule per 250–300 bp of supercoiled DNA and approximately one StpA molecule per 60–100 bp on linear DNA for strong inhibition of the nucleases. These results support the role of StpA as a nucleoid-structuring protein which binds DNA segments together. The inhibition of EcoKI, which cleaves DNA at a site remote from its initial target sequence after extensive DNA translocation driven by ATP hydrolysis, suggests that these enzymes would be unable to function on chromosomal DNA even during times of DNA damage when potentially lethal, unmodified target sites occur on the chromosome. This supports a role for nucleoid-associated proteins in restriction alleviation during times of cell stress

Strong physical constraints on sequence-specific target location by proteins on DNA molecules

Sequence-specific binding to DNA in the presence of competing non-sequence-specific ligands is a problem faced by proteins in all organisms. It is akin to the problem of parking a truck at a loading bay by the side of a road in the presence of cars parked at random along the road. Cars even partially covering the loading bay prevent correct parking of the truck. Similarly on DNA, non-specific ligands interfere with the binding and function of sequence-specific proteins. We derive a formula for the probability that the loading bay is free from parked cars. The probability depends on the size of the loading bay and allows an estimation of the size of the footprint on the DNA of the sequence-specific protein by assaying protein binding or function in the presence of increasing concentrations of non-specific ligand. Assaying for function gives an ‘activity footprint’; the minimum length of DNA required for function rather than the more commonly measured physical footprint. Assaying the complex type I restriction enzyme, EcoKI, gives an activity footprint of ∼66 bp for ATP hydrolysis and 300 bp for the DNA cleavage function which is intimately linked with translocation of DNA by EcoKI. Furthermore, considering the coverage of chromosomal DNA by proteins in vivo, our theory shows that the search for a specific DNA sequence is very difficult; most sites are obscured by parked cars. This effectively rules out any significant role in target location for mechanisms invoking one-dimensional, linear diffusion along DNA

Spaces for play:Listening to children's voices

This paper explores young children’s voices about their play spaces in one Scottish primary school. 45 children (ages 5-7 years) participated, choosing from a range of creative methods (e.g., InPhoTours, drawing, mapping) to share their voices. Using a ‘Playful Research Ethics Framework’, a developmentally appropriate framework which involved the use of visual aids, puppets, songs, Makaton symbols and discussions as well as attention to any cues of disengagement, this research aimed at achieving children’s ongoing informed assent. Four themes were identified: (a) the ‘whole child’ in the space, (b) space and relationships, (c) function of space, and (d) impact (or lack) of children’s voices about space. Children expressed differences in ownership, creativity and imagination in indoor and outdoor spaces. Further, despite perceiving there to be a lack of agency, children were willing to share their voices. There are implications for both practice and research in terms of adults willing to effectively listen to children’s voices and acting on them. This study makes original and significant contributions which have the potential to impact research and practice with young children internationally

Influence of Type I Fimbriae and Fluid Shear Stress on Bacterial Behavior and Multicellular Architecture of Early Escherichia coli Biofilms at Single-Cell Resolution.

Biofilm formation on abiotic surfaces in the food and medical industry can cause severe contamination and infection, yet how biological and physical factors determine the cellular architecture of early biofilms and the bacterial behavior of the constituent cells remains largely unknown. In this study, we examined the specific role of type I fimbriae in nascent stages of biofilm formation and the response of microcolonies to environmental flow shear at the single-cell resolution. The results show that type I fimbriae are not required for reversible adhesion from plankton, but they are critical for the irreversible adhesion of Escherichia coli strain MG1655 cells that form biofilms on polyethylene terephthalate (PET) surfaces. Besides establishing firm cell surface contact, the irreversible adhesion seems necessary to initiate the proliferation of E. coli on the surface. After the application of shear stress, bacterial retention is dominated by the three-dimensional architecture of colonies, independent of the population size, and the multilayered structure could protect the embedded cells from being insulted by fluid shear, while the cell membrane permeability mainly depends on the biofilm population size and the duration of the shear stress.IMPORTANCE Bacterial biofilms could lead to severe contamination problems in medical devices and food processing equipment. However, biofilms are usually studied at a rough macroscopic level; thus, little is known about how individual bacterium behavior within biofilms and the multicellular architecture are influenced by bacterial appendages (e.g., pili/fimbriae) and environmental factors during early biofilm formation. We applied confocal laser scanning microscopy (CLSM) to visualize Escherichia coli microcolonies at a single-cell resolution. Our findings suggest that type I fimbriae are vital to the initiation of bacterial proliferation on surfaces. We also found that the fluid shear stress affects the biofilm architecture and cell membrane permeability of the constituent bacteria in a different way: the onset of the biofilm is linked with the three-dimensional morphology, while membranes are regulated by the overall population of microcolonies

Quantitative evaluation of degenerated tendon model using combined optical coherence elastography and acoustic radiation force method

Damage of collagen fibers in tendons is often directly related to changes in a tendon’s mechanical properties. Direct quantitative elasticity measurement of tendons will provide important information in tendon dysfunction diagnosis and treatment assessment. A feasibility study of quantifying the mechanical properties of a degenerated tendon model by a nondestructive imaging modality, which combines optical coherence elastography and acoustic radiation force (ARF) method, is presented. The degenerated tendon model was produced by the partial degradation of chicken tendons through incubation with collagenase at different concentrations and incubation times. A 30-kHz longitudinal ultrasound transducer was used to provide an ARF signal, which was detected by an ultra-high sensitive phase sensitive optical coherence tomography (PhS-OCT) system. The experimental results demonstrate that the combination of ARF method and PhS-OCT can measure the elasticity of tendon quantitatively. The corresponding changes in tendon elasticity due to the application of collagenase have been revealed by this new imaging modality. This method can potentially be used in the assessment of tissue engineering products and in the diagnosis and treatment progression of tendon diseases