Evidence of Stage Shift in Women Diagnosed With Ovarian Cancer During Phase II of the United Kingdom Familial Ovarian Cancer Screening Study.

Purpose To establish the performance of screening with serum cancer antigen 125 (CA-125), interpreted using the risk of ovarian cancer algorithm (ROCA), and transvaginal sonography (TVS) for women at high risk of ovarian cancer (OC) or fallopian tube cancer (FTC). Patients and Methods Women whose estimated lifetime risk of OC/FTC was ≥ 10% were recruited at 42 centers in the United Kingdom and underwent ROCA screening every 4 months. TVS occurred annually if ROCA results were normal or within 2 months of an abnormal ROCA result. Risk-reducing salpingo-oophorectomy (RRSO) was encouraged throughout the study. Participants were observed via cancer registries, questionnaires, and notification by centers. Performance was calculated after censoring 365 days after prior screen, with modeling of occult cancers detected at RRSO. Results Between June 14, 2007, and May 15, 2012, 4,348 women underwent 13,728 women-years of screening. The median follow-up time was 4.8 years. Nineteen patients were diagnosed with invasive OC/FTC within 1 year of prior screening (13 diagnoses were screen-detected and six were occult at RRSO). No symptomatic interval cancers occurred. Ten (52.6%) of the total 19 diagnoses were stage I to II OC/FTC (CI, 28.9% to 75.6%). Of the 13 screen-detected cancers, five (38.5%) were stage I to II (CI, 13.9% to 68.4%). Of the six occult cancers, five (83.3%) were stage I to II (CI, 35.9% to 99.6%). Modeled sensitivity, positive predictive value, and negative predictive value for OC/FTC detection within 1 year were 94.7% (CI, 74.0% to 99.9%), 10.8% (6.5% to 16.5%), and 100% (CI, 100% to 100%), respectively. Seven (36.8%) of the 19 cancers diagnosed < 1 year after prior screen were stage IIIb to IV (CI, 16.3% to 61.6%) compared with 17 (94.4%) of 18 cancers diagnosed > 1 year after screening ended (CI, 72.7% to 99.9%; P < .001). Eighteen (94.8%) of 19 cancers diagnosed < 1 year after prior screen had zero residual disease (with lower surgical complexity, P = .16) (CI, 74.0% to 99.9%) compared with 13 (72.2%) of 18 cancers subsequently diagnosed (CI, 46.5% to 90.3%; P = .09). Conclusion ROCA-based screening is an option for women at high risk of OC/FTC who defer or decline RRSO, given its high sensitivity and significant stage shift. However, it remains unknown whether this strategy would improve survival in screened high-risk women.

Surface modification of graphene nanopores for protein translocation.

Studies of DNA translocation through graphene nanopores have revealed their potential for DNA sequencing. Here we report a study of protein translocation through chemically modified graphene nanopores. A transmission electron microscope (TEM) was used to cut nanopores with diameters between 5 and 20 nm in multilayer graphene prepared by chemical vapor deposition (CVD). After oxygen plasma treatment, the dependence of the measured ionic current on salt concentration and pH was consistent with a small surface charge induced by the formation of carboxyl groups. While translocation of gold nanoparticles (10 nm) was readily detected through such treated pores of a larger diameter, translocation of the protein ferritin was not observed either for oxygen plasma treated pores, or for pores modified with mercaptohexadecanoic acid. Ferritin translocation events were reliably observed after the pores were modified with the phospholipid-PEG (DPPE-PEG750) amphiphile. The ion current signature of translocation events was complex, suggesting that a series of interactions between the protein and pores occurs during the process.

Mass transport through vertically aligned large diameter MWCNTs embedded in parylene.

We have fabricated porous membranes using a parylene encapsulated vertically aligned forest of multi-walled carbon nanotubes (MWCNTs, about 7 nm inner diameter). The transport of charged particles in electrolyte through these membranes was studied by applying electric field and pressure. Under an electric field in the range of 4.4 × 10(4) V m(-1), electrophoresis instead of electroomosis is found to be the main mechanism for ion transport. Small molecules and 5 nm gold nanoparticles can be driven through the membranes by an electric field. However, small biomolecules, like DNA oligomers, cannot. Due to the weak electric driving force, the interactions between charged particles and the hydrophobic CNT inner surface play important roles in the transport, leading to enhanced selectivity for small molecules. Simple chemical modification on the CNT ends also induces an obvious effect on the translocation of single strand DNA oligomers and gold nanoparticles under a modest pressure (<294 Pa).

Phylogenetic analysis of cryptic speciation in the polychaete Pygospio elegans.

Development in marine invertebrate species can take place through a variety of modes and larval forms, but within a species, developmental mode is typically uniform. Poecilogony refers to the presence of more than one mode of development within a single species. True poecilogony is rare, however, and in some cases, apparent poecilogony is actually the result of variation in development mode among recently diverged cryptic species. We used a phylogenetic approach to examine whether poecilogony in the marine polychaete worm, Pygospio elegans, is the result of cryptic speciation. Populations of worms identified as P. elegansooded, and intermediate larvae; these modes are found both within and among populations. We examined sequence variation among partial mitochondrial cytochrome c oxidase subunit I sequences obtained for 279 individual worms sampled across broad geographic and environmental scales. Despite a large number of unique haplotypes (121 haplotypes from 279 individuals), sequence divergence among European samples was low (1.7%) with most of the sequence variation observed within populations, relative to the variation among regions. More importantly, we observed common haplotypes that were widespread among the populations we sampled, and the two most common haplotypes were shared between populations differing in developmental mode. Thus, our results support an earlier conclusion of poecilogony in P elegans. In addition, predominantly planktonic populations had a larger number of population-specific low-frequency haplotypes. This finding is largely consistent with interspecies comparisons showing high diversity for species with planktonic developmental modes in contrast to low diversity in species with brooded developmental modes.

Electronic sensitivity of a single-walled carbon nanotube to internal electrolyte composition.

Carbon nanotubes (CNTs) are well known as materials for nanoelectronics and show great potential to be used as the sensing elements in chemical and biological sensors. Recently, CNTs have been shown to be effective nanofluidic channels and the transport of substances through small diameter CNTs is intrinsically fast, selective, and operates at the single molecule level. It has been shown that the transport characteristics of semiconducting single-walled CNT (SWCNT) field effect transistors (FETs) are sensitive to internal water wetting. We report here that the characteristics of semiconducting SWCNT FETs are also sensitive to the concentration, pH and ion type of the ionic solution when the electrolyte is inside the CNT. Such sensitivity is not observed at the outside surface of a semiconducting SWCNT. This opens a new avenue for building new types of CNT sensor devices in which the SWCNT concurrently functions as a nanochannel and an electronic detector.

A hydrogen-bonded electron-tunneling circuit reads the base composition of unmodified DNA.

Using a tunnel junction in which one electrode is guanidinium-functionalized (to trap DNA via hydrogen bonding to the backbone phosphates) and a second electrode which is functionalized with a base (to capture its complementary target on the DNA), current versus distance curves are obtained which yield an accurate measure of the base composition of DNA oligomers. With this long tunneling path, resolution is limited to sequence blocks of about twenty bases or larger, because of the need to form a large-area tunnel junction. A shorter hydrogen-bonded path across bases will be required for DNA sequencing. Nonetheless, these measurements point the way to a new type of nanoscale sensor.

Can an atomic force microscope sequence DNA using a nanopore?

R. Bension has proposed that single molecules of DNA could be sequenced rapidly, in long sequential reads, by reading off the force required to pull a tightly fitting molecular ring over each base in turn using an atomic force microscope (AFM). We present molecular dynamics simulations that indicate that pulling DNA very rapidly (m/s) could generate large force peaks as each base is passed ( approximately 1 nN) with significant differences ( approximately 0.5 nN) between purine and pyrimidine. These speeds are six orders of magnitude faster than could be read out by a conventional AFM, and extending the calculations to accessible speeds using Kramers' theory shows that thermal fluctuations dominate the process with the result that purine and pyrimidine cannot be distinguished with the pulling speeds attained by current AFM technology.

AFM imaging of protein movements: histone H2A-H2B release during nucleosome remodeling.

Being able to follow assembly/disassembly reactions of biomolecular complexes directly at the single molecule level would be very useful. Here, we use an AFM technique that can simultaneously obtain topographic images and identify the locations of a specific type of protein within those images to monitor the histone H2A component of nucleosomes acted on by human Swi-Snf, an ATP-dependent nucleosome remodeling complex. Activation of remodeling results in significant H2A release from nucleosomes, based on recognition imaging and nucleosome height changes, and changes in the recognition patterns of H2A associated directly with hSwi-Snf complexes.

Isolation of an scFv targeting BRG1 using phage display with characterization by AFM.

Remodeling of chromatin is a vitally important event in processes such as transcription and replication. Brahma-related gene 1 (BRG1) protein is the major ATPase subunit in the human Swi/Snf complex (hSwi/Snf), an important example of the family of enzymes that carry out such remodeling events. We have used a recently developed technique, recognition imaging, to better understand the role of BRG1 in remodeling chromatin. In such experiments, a specific antibody against BRG1 is needed. However, we have found that the commercially available polyclonal (CAP) antibodies interact non-specifically with nucleosomes, making it impossible to identify hSwi/Snf (BRG1) in their presence. Here antibody phage display technology is employed for development of an antibody specifically targeting BRG1. The Tomlinson I and J single chain variable fragment (scFv) libraries were used for successful isolation of an anti-BRG1 scFv. We demonstrate that the scFv binds more strongly and with less nonspecific interactions than the CAP antibody. This work lays the groundwork for future studies involving chromatin remodeling.

Solution AFM studies of human Swi-Snf and its interactions with MMTV DNA and chromatin.

ATP-dependent nucleosome remodeling complexes are crucial for relieving nucleosome repression during transcription, DNA replication, recombination, and repair. Remodeling complexes can carry out a variety of reactions on chromatin substrates but precisely how they do so remains a topic of active inquiry. Here, a novel recognition atomic force microscopy (AFM) approach is used to characterize human Swi-Snf (hSwi-Snf) nucleosome remodeling complexes in solution. This information is then used to locate hSwi-Snf complexes bound to mouse mammary tumor virus promoter nucleosomal arrays, a natural target of hSwi-Snf action, in solution topographic AFM images of surface-tethered arrays. By comparing the same individual chromatin arrays before and after hSwi-Snf activation, remodeling events on these arrays can be monitored in relation to the complexes bound to them. Remodeling is observed to be: inherently heterogeneous; nonprocessive; able to occur near and far from bound complexes; often associated with nucleosome height decreases. These height decreases frequently occur near sites of DNA release from chromatin. hSwi-Snf is usually incorporated into nucleosomal arrays, with multiple DNA strands entering into it from various directions, + or - ATP; these DNA paths can change after hSwi-Snf activation. hSwi-Snf appears to interact with naked mouse mammary tumor virus DNA somewhat differently than with chromatin and ATP activation of surface-bound DNA/hSwi-Snf produces no changes detectable by AFM.

Screening for abdominal aortic aneurysm in a geographically isolated area.

BACKGROUND: Screening for abdominal aortic aneurysm has been shown to reduce aneurysm-related mortality, but the applicability of the results to the whole of the UK has been questioned. This study examined screening in a remote and rural area. METHODS: Over 3 years, men aged 65-74 years were offered screening in the community by ultrasonography, usually in general practitioner surgeries. Men with an aneurysm were rescanned at intervals or assessed for surgery. The screening and hospital costs of the programme were calculated. RESULTS: Some 9323 men were offered screening of whom 8355 (89.6 per cent) attended. Uptake was high in all areas. A total of 430 scans (5.1 per cent) were abnormal; 40 men had an aneurysm greater than 55 mm in diameter. Twenty further men had an aorta that enlarged to greater than 55 mm during follow-up. A total of 54 men had elective repair with one death (mortality rate 2 per cent). The cost of screening alone was 16 pound per invitation and the overall cost of the programme, including surgery, was 58 pound per invitation. CONCLUSION: Screening for abdominal aortic aneurysm can be carried out in a remote and rural area with high uptake, acceptable clinical results and at no greater cost than in more densely populated areas.

A statistical thermodynamic model applied to experimental AFM population and location data is able to quantify DNA-histone binding strength and internucleosomal interaction differences between acetylated and unacetylated nucleosomal arrays.

Imaging of nucleosomal arrays by atomic force microscopy allows a determination of the exact statistical distributions for the numbers of nucleosomes per array and the locations of nucleosomes on the arrays. This precision makes such data an excellent reference for testing models of nucleosome occupation on multisite DNA templates. The approach presented here uses a simple statistical thermodynamic model to calculate theoretical population and positional distributions and compares them to experimental distributions previously determined for 5S rDNA nucleosomal arrays (208-12,172-12). The model considers the possible locations of nucleosomes on the template, and takes as principal parameters an average free energy of interaction between histone octamers and DNA, and an average wrapping length of DNA around the octamers. Analysis of positional statistics shows that it is possible to consider interactions between nucleosomes and positioning effects as perturbations on a random positioning noninteracting model. Analysis of the population statistics is used to determine histone-DNA association constants and to test for differences in the free energies of nucleosome formation with different types of histone octamers, namely acetylated or unacetylated, and different DNA templates, namely 172-12 or 208-12 5S rDNA multisite templates. The results show that the two template DNAs bind histones with similar affinities but histone acetylation weakens the association of histones with both templates. Analysis of locational statistics is used to determine the strength of specific nucleosome positioning tendencies by the DNA templates, and the strength of the interactions between neighboring nucleosomes. The results show only weak positioning tendencies and that unacetylated nucleosomes interact much more strongly with one another than acetylated nucleosomes; in fact acetylation appears to induce a small anticooperative occupation effect between neighboring nucleosomes.

Using atomic force microscopy to study nucleosome remodeling on individual nucleosomal arrays in situ.

In eukaryotes, genomic processes like transcription, replication, repair, and recombination typically require alterations in nucleosome structure on specific DNA regions to operate. ATP-dependent nucleosome remodeling complexes provide a major mechanism for carrying out such alterations in vivo. To learn more about the action of these important complexes, we have utilized an atomic force microscopy in situ technique that permits comparison of the same individual molecules before and after activation of a particular process, in this case nucleosome remodeling. This direct approach was used to look for changes induced by the action of the human Swi-Snf remodeling complex on individual, single-copy mouse mammary tumor virus promoter nucleosomal arrays. Using this technique, we detect a variety of changes on remodeling. Many of these changes are larger in scale than suggested from previous studies and involve a number of DNA-mediated events, including a preference for the removal of a complete turn (80 basepairs) of nucleosomal DNA. The latter result raises the possibility of an unanticipated mode of human Swi-Snf interaction with the nucleosome, namely via the 11-nm histone surface.

Single-molecule recognition imaging microscopy.

Atomic force microscopy is a powerful and widely used imaging technique that can visualize single molecules and follow processes at the single-molecule level both in air and in solution. For maximum usefulness in biological applications, atomic force microscopy needs to be able to identify specific types of molecules in an image, much as fluorescent tags do for optical microscopy. The results presented here demonstrate that the highly specific antibody-antigen interaction can be used to generate single-molecule maps of specific types of molecules in a compositionally complex sample while simultaneously carrying out high-resolution topographic imaging. Because it can identify specific components, the technique can be used to map composition over an image and to detect compositional changes occurring during a process.

Nucleosomal arrays can be salt-reconstituted on a single-copy MMTV promoter DNA template: their properties differ in several ways from those of comparable 5S concatameric arrays.

Subsaturated nucleosomal arrays were reconstituted on a single-copy MMTV promoter DNA fragment by salt dialysis procedures and studied by atomic force microscopy. Up to an occupation level of approximately eight nucleosomes on this 1900 bp template, salt reconstitution produces nucleosomal arrays which look very similar to comparably loaded 5S rDNA nucleosomal arrays; i.e., nucleosomes are dispersed on the DNA template. Thus, at these occupation levels, the single-copy MMTV template forms arrays suitable for biophysical analyses. A quantitative comparison of the population features of subsaturated MMTV and 5S arrays detects differences between the two: a requirement for higher histone levels to achieve a given level of nucleosome occupation on MMTV templates, indicating that nucleosome loading is thermodynamically less favorable on this template; a preference for pairwise nucleosome occupation of the MMTV (but not the 5S) template at midrange occupation levels; and an enhanced salt stability for nucleosomes on MMTV versus 5S arrays, particularly in the midrange of array occupation. When average occupation levels exceed approximately eight nucleosomes per template, MMTV arrays show a significant level of mainly intramolecular compaction; 5S arrays do not. Taken together, these results show clearly that the nature of the underlying DNA template can affect the physical properties of nucleosomal arrays. DNA sequence-directed differences in the physical properties of chromatin may have important consequences for functional processes such as gene regulation.

Bias-induced forces in conducting atomic force microscopy and contact charging of organic monolayers.

Contact electrification, a surface property of bulk dielectric materials, has now been observed at the molecular scale using conducting atomic force microscopy (AFM). Conducting AFM measures the electrical properties of an organic film sandwiched between a conducting probe and a conducting substrate. This paper describes physical changes in the film caused by the application of a bias. Contact of the probe leads to direct mechanical stress and the applied electric field results in both Maxwell stresses and electrostriction. Additional forces arise from charge injection (contact charging). Electrostriction and contact charging act oppositely from the normal long-range Coulomb attraction and dominate when a charged tip touches an insulating film, causing the tip to deflect away from the film at high bias. A bias-induced repulsion observed in spin-coated PMMA films may be accounted for by either mechanism. In self-assembled monolayers, however, tunnel current signals show that the repulsion is dominated by contact charging.

Reproducible measurement of single-molecule conductivity.

A reliable method has been developed for making through-bond electrical contacts to molecules. Current-voltage curves are quantized as integer multiples of one fundamental curve, an observation used to identify single-molecule contacts. The resistance of a single octanedithiol molecule was 900 +/- 50 megohms, based on measurements on more than 1000 single molecules. In contrast, nonbonded contacts to octanethiol monolayers were at least four orders of magnitude more resistive, less reproducible, and had a different voltage dependence, demonstrating that the measurement of intrinsic molecular properties requires chemically bonded contacts.

Conformation and rigidity of DNA microcircles containing waf1 response element for p53 regulatory protein.

The tumor-suppressor activity of p53 is closely related to its DNA-binding properties. It binds a number of DNA response-elements and it is likely that these share a common structural feature. Here, we present a new, general method to determine the absolute twist of flexible DNA promoter sequences based on direct imaging of the topology of microcircles containing the sequences. We have used magnetically driven dynamic force microscopy ("MacMode" AFM) to observe, in solution, the conformation of 168 base-pair DNA microcircles, each containing four equally spaced copies of the waf1/cip1/p21 p53 response-element. Analysis of the images showed that the microcircles are markedly puckered with a small excess of negatively writhed molecules. The average measured values of writhe are 0.109+/-0.013 (for 60 positively writhed molecules) and -0.098+/-0.011 (for 65 negatively writhed molecules). These values lead directly to a difference in linking number for the positively and negatively writhed molecules prior to ligation, from which we derive a twist mismatch of 178 degrees (overtwist). This is 44.5 degrees for each 42-mer precursor containing a single waf1/cip1/p21 p53 response-element, in good agreement with the range of values deduced by indirect biochemical techniques. The two values of writhe may also be used to determine the ratio of the bending (B) to twisting (C) rigidity, yielding B/C=0.23. This is about one-third of the value for long, random-sequence DNA, suggesting that the waf1/cip1/p21 p53 response-element is extremely flexible, a result that is also consistent with indirect biochemical experiments. These results support the idea, proposed by us earlier, that torsional stress may play a role in the regulation of p53 binding through modulation of twist at the binding site.

Single molecule force spectroscopy in biology using the atomic force microscope.

The importance of forces in biology has been recognized for quite a while but only in the past decade have we acquired instrumentation and methodology to directly measure interactive forces at the level of single biological macromolecules and/or their complexes. This review focuses on force measurements performed with the atomic force microscope. A general introduction to the principle of action is followed by review of the types of interactions being studied, describing the main results and discussing the biological implications.

Conformational transition in DNA on a cold surface.

The contour length of DNA fragments, deposited and imaged on mica under buffer, was measured as a function of deposition temperature. Extended DNA molecules (on Ni- and silane-treated surfaces) contract rapidly with falling temperature, approaching the contour length of A-DNA at 2 degrees C. The contraction is not unique to a specific sequence and does not occur in solution at 2 degrees C or on a surface at 25 degrees C, indicating that it arises from a combination of low temperature and surface contact. It is probably a consequence of reduced water activity at a cold surface.