Mechanics and Function of DNA Looping and Supercoiling.

DNA is an essential molecule that enables the storage and retrieval of genetic information. Since the discovery of its structure (double helix), the relationship between the molecule's structure and function has been studied extensively. Here we extend beyond the static structure and consider how the mechanical properties and dynamics influence its function. To do so, we exercise an elasto-dynamic rod model for DNA. By exercising this model, we study two biologically relevant systems. First, we study DNA looping by Lac repressor. Although this is a classic gene regulatory system, the mechanics of the DNA loop remain largely unknown. Therefore, we compute the effects of inter-operator length, intrinsic curvature, and protein flexibility on the energetics and topology these loops. We calculate that anti-parallel loops are energetically preferred, the elastic energy of a family of intrinsically curved DNA loops spans 5-12 kT, and identify the sensitivity of elastic energy to protein flexibility. Our computations compare favorably with published experimental data and motivate experimental work in the Kahn lab at the University of Maryland. Furthermore, we contribute an efficient method to analyze a large family of intrinsically curved DNA molecules and a method to account for Lac repressor flexibility in our rod model. In addition, we analyze cryo-EM images (obtained by the Stasiak lab at the Université de Lausanne) of DNA minicircles with similar lengths to the Lac repressor DNA loops. Second, we study the relaxation of DNA supercoils by topoisomerase. In doing so, we make advancements to the rod model and perform the first multi-scale model of supercoil relaxation by topoisomerase. Specifically, we contribute an efficient method to account for self contact and electrostatics in our elastic rod model. In our multi-scale simulation we couple our rod model with recent data (from MD simulations by the Andricioaei lab at the University of California - Irvine) that characterizes the the mechanics of topoisomerase. In doing so we gain insight into the dynamics of supercoil relaxation and make a first prediction of the relaxation time (0.1-1.0 μs).Ph.D.Mechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/75861/1/tlillian_1.pd

Ethnicity or cultural group identity of pregnant women in Sydney, Australia: is country of birth a reliable proxy measure?

Background: Australia has one of the most ethnically and culturally diverse maternal populations in the world. Routinely few variables are recorded in clinical data or health research to capture this diversity. This paper explores and how pregnant women, Australian-born and overseas-born, respond to survey questions on ethnicity or a cultural group identity, and whether country of birth is a reliable proxy measure. Methods: Frequency tabulations and inductive qualitative analysis of data from two questions on country of birth, and identification with an ethnicity or cultural group from a larger survey of pregnant women attending public antenatal clinics across four hospitals in Sydney, Australia. Results: Responses varied widely among the 762 with 75 individual cultural groups or ethnicities and 68 countries of birth reported. For Australian-born women (n=293), 23% identified with a cultural group or ethnicity, and 77% did not. For overseas-born women (n=469), 44% identified with a cultural group or ethnicity and 56% did not. Responses were coded under five emerging themes. Conclusions: Ethnicity and cultural group identity are complex concepts; women across and within countries of birth identified differently. Over three quarters of Australian-born, and over half of over-seas born women, reported no ethnicity or cultural group identity, indicating country of birth is not a reliable measure for identifying diversity. Researchers should scrutinise research questions and data usage, policy makers consider the complexity of ethnicity or cultural group identity, and the limitations of a single variable measure to identify ethnically and culturally diverse pregnant women and deliver woman-centred care.NHMR

Women's views about the timing of birth

Background: Estimated date of birth (EDB) is used to guide clinical management of women during pregnancy and birth, although its imprecision is recognised. Alternatives to the EDB have been suggested for use with women however their attitudes to timing of birth information have not been examined. Aims: To explore women’s expectations of giving birth on or near their EDB, and their attitudes to alternative estimates for timing of birth. Methods: A survey of pregnant women attending four public hospitals in Sydney, Australia, between July and December 2012. Results: Among 769 surveyed women, 42% expected to birth before their due date, 16% after the due date, 15% within a day or so of the due date, and 27% had no expectations. Nulliparous women were more likely to expect to give birth before their due date. Women in the earlier stages of pregnancy were more likely to have no expectations or to expect to birth before the EDB while women in later pregnancy were more likely to expect birth after their due date. For timing of birth information, only 30% of women preferred an EDB; the remainder favoured other options. Conclusions: Most women understood the EDB is imprecise. The majority of women expressed a preference for timing of birth information in a format other than an EDB. In support of woman-centred care, it may be helpful to ask each woman how she would like to receive estimated timing of birth information.NHMR

Cooperative kinking at distant sites in mechanically stressed DNA

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DN

FRET studies of a landscape of Lac repressor-mediated DNA loops

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-β-d-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility

Implementation of the StandingTall programme to prevent falls in older people:a process evaluation protocol

INTRODUCTION: One in three people aged 65 years and over fall each year. The health, economic and personal impact of falls will grow substantially in the coming years due to population ageing. Developing and implementing cost-effective strategies to prevent falls and mobility problems among older people is therefore an urgent public health challenge. StandingTall is a low-cost, unsupervised, home-based balance exercise programme delivered through a computer or tablet. StandingTall has a simple user-interface that incorporates physical and behavioural elements designed to promote compliance. A large randomised controlled trial in 503 community-dwelling older people has shown that StandingTall is safe, has high adherence rates and is effective in improving balance and reducing falls. The current project targets a major need for older people and will address the final steps needed to scale this innovative technology for widespread use by older people across Australia and internationally. METHODS AND ANALYSIS: This project will endeavour to recruit 300 participants across three sites in Australia and 100 participants in the UK. The aim of the study is to evaluate the implementation of StandingTall into the community and health service settings in Australia and the UK. The nested process evaluation will use both quantitative and qualitative methods to explore uptake and acceptability of the StandingTall programme and associated resources. The primary outcome is participant adherence to the StandingTall programme over 6 months. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the South East Sydney Local Health District Human Research Ethics Committee (HREC reference 18/288) in Australia and the North West- Greater Manchester South Research Ethics Committee (IRAS ID: 268954) in the UK. Dissemination will be via publications, conferences, newsletter articles, social media, talks to clinicians and consumers and meetings with health departments/managers. TRIAL REGISTRATION NUMBER: ACTRN12619001329156

Cooperative kinking at distant sites in mechanically stressed DNA

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA