Stability, resolution, and ultra-low wear amplitude modulation atomic force microscopy of DNA: Small amplitude small set-point imaging

A way to operate fundamental mode amplitude modulation atomic force microscopy is introduced which optimizes stability and resolution for a given tip size and shows negligible tip wear over extended time periods (∼24 h). In small amplitude small set-point (SASS) imaging, the cantilever oscillates with sub-nanometer amplitudes in the proximity of the sample, without the requirement of using large drive forces, as the dynamics smoothly lead the tip to the surface through the water layer. SASS is demonstrated on single molecules of double-stranded DNA in ambient conditions where sharp silicon tips (R ∼ 2-5 nm) can resolve the right-handed double helix

Patchiness of ion-exchanged mica revealed by DNA binding dynamics at short length scales

The binding of double-stranded (ds) DNA to mica can be controlled through ion-exchanging the mica with divalent cations. Measurements of the end-to-end distance of linear DNA molecules discriminate whether the binding mechanism occurs through 2D surface equilibration or kinetic trapping. A range of linear dsDNA fragments have been used to investigate length dependences of binding. Mica, ion-exchanged with Ni(II) usually gives rise to kinetically trapped DNA molecules, however, short linear fragments (<800 bp) are seen to deviate from the expected behaviour. This indicates that ion-exchanged mica is heterogeneous, and contains patches or domains, separating different ionic species. These results correlate with imaging of dsDNA under aqueous buffer on Ni(II)-mica and indicate that binding domains are of the order of 100 nm in diameter. Shorter DNA fragments behave intermediate to the two extreme cases of 2D equilibration and kinetic trapping. Increasing the incubation time of Ni(II) on mica, from minutes to hours, brings the conformations of the shorter DNA fragments closer to the theoretical value for kinetic trapping, indicating that long timescale kinetics play a role in ion-exchange. X-ray photoelectron spectroscopy (XPS) was used to confirm that the relative abundance of Ni(II) ions on the mica surface increases with time. These findings can be used to enhance spatial control of binding of DNA to inorganic surfaces with a view to patterning high densities arrays

Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP > Heparin or HepS > DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned λ(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after ‘collision’ has caused their arrest

Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

How does proline-repeat motif length in the proteins of teeth and bones relate to the evolution of vertebrates? Counterintuitively, longer repeat stretches are associated with smaller aggregated subunits within a supramolecular matrix, resulting in enhanced crystal length in mammalian versus amphibian tooth enamel

Oral Antimicrobial Peptides and Biological Control of Caries

The presence of antimicrobial peptides (AMPs) in saliva may be a biological factor that contributes to susceptibility or resistance to caries. This manuscript will review AMPs in saliva, consider their antimicrobial and immunomodulatory functions, and evaluate their potential role in the oral cavity for protection of the tooth surface as well as the oral mucosa. These AMPs are made in salivary gland and duct cells and have broad antimicrobial activity. Alpha-defensins and LL37 are also released by neutrophils into the gingival crevicular fluid. Both sources may account for their presence in saliva. A recent study in middle school children aimed to determine a possible correlation between caries prevalence in children and salivary concentrations of the antimicrobial peptides human beta-defensin-3 (hBD-3), the cathelicidin, LL37, and the alpha-defensins. The levels of these AMPs were highly variable in the population. While levels of LL37 and hBD-3 did not correlate with caries experience, the mean alpha-defensin level was significantly higher in children with no caries than in children with caries (p < 0.005). We conclude that several types of AMPs that may have a role in oral health are present in unstimulated saliva. Low salivary levels of alpha-defensin may represent a biological factor that contributes to caries susceptibility. Our observation could lead to new ways to prevent caries and to a new tool for caries risk assessment

Dataset associated with the "Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model system using ex-situ atomic force microscopy"

Dataset associated with the publication "Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model system using ex-situ atomic force microscopy". The dataset includes the raw AFM images and statistical analysis. It is divided into six parts associated with the following AFM imaging data collected on: 1) cleaved mica; 2) polysaccharide heparin deposited on mica; 3) polysaccharide heparan sulphate deposited on mica; 4) the DNA template with and without the polysaccharides; 5) RNA polymerase with and without the polysaccharides; 6) Open promoter complexes with and without the polysaccharides

A qualitative systematic review of family caregivers’ experiences of artificial nutrition and hydration at home: A meta-ethnography

Background: Artificial nutrition and hydration (AN&H) may be provided toindividuals in the home environment, and family caregivers are often involved in the management of this intervention. This experience can have multiple consequences for families. Aims: The aim of this meta-ethnography is to explore and synthesize the personal experiences of family caregivers providing care to a person receiving home AN&H. Methods & Procedures: A comprehensive search of the literature was conducted without any time limitations applied. Seven stages of meta-ethnography were followed. Public and patient involvement was incorporated into the development of the line of argument synthesis in this review. This review is reported following the eMERGe guidelines and it was registered in PROSPERO. Main Contribution: A total of 22 studies were included representing the experiences of 336 family caregivers. Two main themes emerged: (1) sink or swim, being thrown in at the deep end; and (2) professional support as a bedrock. The first theme represents the experiences from the very start of home AN&H when the family caregivers may be overwhelmed with the level of skills they have to acquire. With time, family caregivers perceived the benefits, but also the challenges, associated with managing home AN&H. If a person receiving home AN&H was able to continue with some oral intake, it had a positive impact on family caregivers’ experiences. The second theme represents the influence of professional support on the lived experience of family caregivers managing home AN&H. This support should be individualized, comprehensive, and co-created with the family caregiver and the person receiving home AN&H. Conclusions & Implications: This review concluded that caring for a person receiving home AN&H can be very challenging for family caregivers. Family caregivers require personalized support from a multidisciplinary team of healthcare professionals to acquire skills, competence and confidence in this new role. Speech and language therapists are important members of this multidisciplinary team because they can facilitate a continuation of oral intake as appropriate.</p