Technical note: Survey of extracellular and cell-pellet-associated DNA from 'touch'/trace samples.

The goal of this study was to characterize the reproducibility of extracellular and cell pellet associated DNA yields recovered from handled substrates. Results showed that extracellular DNA yields were extremely variable between contributors-ranging between 0 and >10ng-and tended to dwarf cell pellet yields, which varied between 0 and ∼230pg. DNA yields across multiple samples from the same contributor on different days showed similar levels of variability in both DNA fractions, indicating that extracellular DNA yield is largely influenced by extrinsic and/or environmental factors and is not a contributor-specific attribute. Microscopic surveys of cells from the pellet fraction as well as fingerprints from the same contributor samples were conducted following treatment with fluorescent DNA stain. Nearly all imaged cells exhibited diffuse fluorescence across the cell without discernable evidence of nuclei. This is consistent with the limited nature of DNA recovery from the pellet fraction and the prevalence of extracellular DNA in these samples.

Open source software tool for the automated detection and characterization of epithelial cells from trace biological samples.

This paper presents a strategy for an unsupervised workflow for identifying epithelial cells in microscopic images and characterizing their morphological and/or optical properties. The proposed method can be used on cells that have been stained with fluorescent dyes and imaged using conventional optical microscopes. The workflow was tested on cell populations that were imaged directly on touch/contact surfaces and stained with nucleic acid dyes to visualize genetic content. Our results show that this approach could be a useful strategy for characterizing differences in staining efficiency and/or morphological properties of individual cells or aggregate populations within a biological sample. Further, they can potentially reduce the laborious nature of microscopic analysis and increase throughput and reproducibility of similar studies.

Nanoscale visualization of extracellular DNA on cell surfaces.

Nanoscale analysis of extracellular DNA (eDNA) that is present on the surface of cells in trace biological samples can provide insight into the understanding of DNA transfer through touch, and thereby, the role of eDNA is a biologically and forensically relevant phenomenon. While various bulk scale tools and DNA analysis can be used to quantitatively obtain this information, obtaining a three dimensional (3D) visualization of the eDNA can provide a unique look into the spatial and temporal dynamics at the cellular level. In this study, we show how atomic force microscopy (AFM) can be integrated with optical microscopy to visualize the distribution of surface associate eDNA at a single cell level. Using a nucleic acid fluorophore such as Diamond™ Dye, the surface eDNA can be observed and quantified using fluorescence microscopy. This informational channel can then be overlaid with surface topography and cellular elasticity to provide structural visualization. Finally, chemical force spectroscopy can be used to obtain the distribution of surface-associated eDNA on the cell surface at the molecular level. Such integrated techniques can enhance understanding of the biological role of eDNA, and can also be potentially valuable for investigating challenging trace samples, containing very few cells for various analyses.

DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle.

Progress in whole-genome sequencing using short-read (e.g., <150 bp), next-generation sequencing technologies has reinvigorated interest in high-resolution physical mapping to fill technical gaps that are not well addressed by sequencing. Here, we report two technical advances in DNA nanotechnology and single-molecule genomics: (1) we describe a labeling technique (CRISPR-Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA and (2) the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM. As a proof of principle, we used this new "nanomapping" method to detect and map precisely BCL2-IGH translocations present in lymph node biopsies of follicular lymphoma patents. This HS-AFM "nanomapping" technique can be complementary to both sequencing and other physical mapping approaches.

High-Speed Atomic Force Microscopy Revealing Contamination in DNA Purification Systems.

Motivated by reports of low-level DNA contamination in popular commercial DNA purification kits, we employed a novel high-speed atomic force microscopy (HS-AFM) method to detect and characterize particulate and polymeric contaminants in four such systems: Qiagen MinElute PCR Purification, Zymo Research DNA Clean and Concentrator-5, Invitrogen ChargeSwitch-Pro PCR Purification, and Beckman Coulter AMPure XP. HS-AFM avoids amplification artifacts present in PCR or in the sequencing of amplified products, and it requires no chemical labels and easily achieves near-single-molecule sensitivity. Using this technique, we found trace levels of filamentous contamination, similar in appearance to dsDNA, in eluates from the Zymo, Qiagen, and ChargeSwitch kits. Conversely, we detected no contaminants in magnetic bead-based AMPure XP solutions. Eluates from the Zymo kits also tested positive for DNA in fluorescent intercalator dye and whole genome amplification (WGA) assays. Qiagen kits tested positive in the fluorescence assay but negative in the WGA assay. Both ChargeSwitch and AMPure XP tested negative in the fluorescence assay while the WGA results for these two kits were ambiguous. Taken together, our findings suggest AMPure XP would be the best choice for analyses requiring very high analytical stringency. While HS-AFM alone does not provide chemical specificity, it is a potentially valuable tool for characterizing and quantifying trace contaminants in molecular biology reagents and instruments in cases where conventional techniques fail.

Atomic force microscopic detection enabling multiplexed low-cycle-number quantitative polymerase chain reaction for biomarker assays.

Quantitative polymerase chain reaction is the current "golden standard" for quantification of nucleic acids; however, its utility is constrained by an inability to easily and reliably detect multiple targets in a single reaction. We have successfully overcome this problem with a novel combination of two widely used approaches: target-specific multiplex amplification with 15 cycles of polymerase chain reaction (PCR), followed by single-molecule detection of amplicons with atomic force microscopy (AFM). In test experiments comparing the relative expression of ten transcripts in two different human total RNA samples, we find good agreement between our single reaction, multiplexed PCR/AFM data, and data from 20 individual singleplex quantitative PCR reactions. This technique can be applied to virtually any analytical problem requiring sensitive measurement concentrations of multiple nucleic acid targets.

Can general practitioners do the follow-ups after surgery with ventilation tubes in the tympanic membrane? Two years audiological data.

BACKGROUND: A university hospital in Mid-Norway has modified their guidelines for follow-up after insertion of ventilation tubes (VTs) in the tympanic membrane, transferring the controls of the healthiest children to general practitioners (GPs). The aim of this study was to evaluate the implementation of these guidelines by exploring audiological outcome and subjective hearing complaints two years after surgery, assessing if follow-ups in general practice resulted in poorer outcome. METHODS: A retrospective observational study was performed at the university hospital and in general practice in Mid-Norway. Children below 18 years who underwent surgery with VTs between Nov 1st 2007 and Dec 31st 2008 (n = 136) were invited to participate. Pure tone audiometry, speech audiometry and tympanometry were measured. A self-report questionnaire assessed subjective hearing, ear complaints and the location of follow-ups. This study includes enough patients to observe group differences in mean threshold (0.5-1-2-4 kHz) of 9 dB or more. RESULTS: There were no preoperative differences in audiometry or tympanometry between the children scheduled for follow-ups by GPs (n = 23) or otolaryngologists (n = 50). Two years after surgery there were no differences between the GP and otolaryngologist groups in improvement of mean hearing thresholds (12.8 vs 12.6 dB, p = 0.9) or reduction of middle ears with effusion (78.0 vs 75.0%, p = 0.9). We found no differences between the groups in terms of parental reports of child hearing or ear complaints. CONCLUSIONS: Implementation of new clinical guidelines for follow-ups after insertion of VTs did not negatively affect audiological outcomes or subjective hearing complaints two years after surgery.

Implementing guidelines for follow-up after surgery with ventilation tube in the tympanic membrane in Norway: a retrospective study.

BACKGROUND: When clinical guidelines are being changed a strategy is required for implementation. St. Olavs University Hospital in Norway modified their guidelines for the follow-up care of children after insertion of ventilation tubes (VT) in the tympanic membrane, transferring the controls of the healthiest children to General Practitioners (GPs). This study evaluates the implementation process in the hospital and in general practice by exploring two issues: 1) Whether the hospital discharged the patients they were supposed to and 2) whether the children consulted a GP for follow-up care. METHODS: A retrospective observational study was performed at St. Olavs University Hospital, Norway and general practice in Mid-Norway. Children under the age of 18 who underwent insertion of VT between Nov 1st 2007 and Dec 31st 2008 (n = 136) were included. Degree of guideline adherence at the hospital and in general practice was measured. RESULTS: The hospital adhered to the guidelines in two-thirds (68.5%) of the patients, planning more patients for follow-up by their GP than recommended in the guidelines (25.8% vs. 12.4%). All except one contacted their GP for control. In total 60% were referred back to specialist health services within two years. CONCLUSIONS: The methods for guideline implementation were successful in securing consultations for follow-up care in general practice. Lack of guideline adherence in the hospital can partly be explained by the lack of quality of the guideline. Further studies are needed to evaluate the quality of controls done by the GPs in order to consider implications for follow-up after VT surgery.

The development of memory for own- and other-race faces.

This study demonstrates that experience and development interact to influence the "cross-race effect." In a multination study (n=245), Caucasian children and adults of European ancestry living in the United States, Norway, or South Africa, as well as biracial (Caucasian-African American) children and adults living in the United States, were tested for recognition of Asian, African, and Caucasian faces. Regardless of national or biracial background, 8- to 10-year-olds, 12- to 14-year-olds, and adults recognized own-race faces more accurately than other-race faces, and did so to a similar extent, whereas 5- to 7-year-olds recognized all face types equally well. This same developmental pattern emerged for biracial children and adults. Thus, early meaningful exposure did not substantially alter the developmental trajectory. During young childhood, developmental influences on face processing operate on a system sufficiently plastic to preclude, under certain conditions, the cross-race effect.