Transverse magnetic tweezers allowing coincident epi-fluorescence microscopy on horizontally extended DNA

Longitudinal magnetic tweezers (L-MT) have seen wide-scale adoption as the tool-of-choice for stretching and twisting a single DNA molecule. They are also used to probe topological changes in DNA as a result of protein binding and enzymatic activity. However, in the longitudinal configuration, the DNA molecule is extended perpendicular to the imaging plane. As a result, it is only possible to infer biological activity from the motion of the tethered superparamagnetic microsphere. Described here is a “transverse” magnetic tweezers (T-MT) geometry featuring simultaneous control of DNA extension and spatially coincident video-rate epi-fluorescence imaging. Unlike in L-MT, DNA tethers in T-MT are extended parallel to the imaging plane between two micron-sized spheres, and importantly protein targets on the DNA can be localised using fluorescent nanoparticles. The T-MT can manipulate a long DNA construct at molecular extensions approaching the contour length defined by B-DNA helical geometry, and the measured entropic elasticity agrees with the worm-like chain model (force < 35 pN). By incorporating a torsionally constrained DNA tether, the T-MT would allow both the relative extension and twist of the tether to be manipulated, while viewing far-red emitting fluorophore-labelled targets. This T-MT design has the potential to enable the study of DNA binding and remodelling processes under conditions of constant force and defined torsional stress

Fabrication of extracellular matrix-derived foams and microcarriers as tissue-specific cell culture and delivery platforms

© 2017 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications

Bovine viral diarrhoea virus infection disrupts uterine interferon stimulated gene regulatory pathways during pregnancy recognition in cows

In cattle, conceptus-derived interferon tau (IFNT) is the pregnancy recognition (PR) signal. Our previous studies showed that non-cytopathic bovine viral diarrhoea virus (ncpBVDV) infection inhibited IFNT-induced interferon stimulated gene (ISG) expression, potentially causing early embryonic death. This study investigated the effect of bovine viral diarrhoea virus (BVDV) infection on upstream regulatory pathways of ISG production using an established PR model. Uterine endometrial cells from 10 apparently healthy and BVDV free cows were cultured and treated with 0 or 100 ng/mL IFNT for 24 h in the presence or absence of ncpBVDV infection. Microarray and pathway analysis were used to determine the IFNT-induced upstream regulators. Expression of the genes associated with the identified pathways were quantified with qPCR. IFNT challenge activated the signalling pathways associated with IFN receptors, JAK1/TYK2, IRFs and STATs and ncpBVDV infection inhibited the activation of IFNT on this pathway. Inhibition of this upstream signalling pathway may thus reduce ISG production to disrupt maternal PR. In addition, the reduction of uterine immunity by ncpBVDV infection may predispose the animals to uterine infection, which in turn impairs their reproductive performance. This provides a mechanism of how BVDV infection leads to early pregnancy failure in cows

Comparing alternating pressure mattresses and high-specification foam mattresses to prevent pressure ulcers in high-risk patients: the PRESSURE 2 RCT

Background: Pressure ulcers (PUs) are a burden to patients, carers and health-care providers. Specialist mattresses minimise the intensity and duration of pressure on vulnerable skin sites in at-risk patients. Primary objective: Time to developing a new PU of category ≥ 2 in patients using an alternating pressure mattress (APM) compared with a high-specification foam mattress (HSFM). Design: A multicentre, Phase III, open, prospective, planned as an adaptive double-triangular group sequential, parallel-group, randomised controlled trial with an a priori sample size of 2954 participants. Randomisation used minimisation (incorporating a random element). Setting: The trial was set in 42 secondary and community inpatient facilities in the UK. Participants: Adult inpatients with evidence of acute illness and at a high risk of PU development. Interventions and follow-up: APM or HSFM – the treatment phase lasted a maximum of 60 days; the final 30 days were post-treatment follow-up. Main outcome measures: Time to event. Results: From August 2013 to November 2016, 2029 participants were randomised to receive either APM (n = 1016) or HSFM (n = 1013). Primary end point – 30-day final follow-up: of the 2029 participants in the intention-to-treat population, 160 (7.9%) developed a new PU of category ≥ 2. There was insufficient evidence of a difference between groups for time to new PU of category ≥ 2 [Fine and Gray model HR 0.76, 95% confidence interval (CI) 0.56 to 1.04; exact p-value of 0.0890 and 2% absolute difference]. Treatment phase sensitivity analysis: 132 (6.5%) participants developed a new PU of category ≥ 2 between randomisation and end of treatment phase. There was a statistically significant difference in the treatment phase time-to-event sensitivity analysis (Fine and Gray model HR 0.66, 95% CI 0.46 to 0.93; p = 0.0176 and 2.6% absolute difference). Secondary end points – 30-day final follow-up: new PUs of category ≥ 1 developed in 350 (17.2%) participants, with no evidence of a difference between mattress groups in time to PU development, (Fine and Gray model HR 0.83, 95% CI 0.67 to 1.02; p-value = 0.0733 and absolute difference 3.1%). New PUs of category ≥ 3 developed in 32 (1.6%) participants with insufficient evidence of a difference between mattress groups in time to PU development (Fine and Gray model HR 0.81, 95% CI 0.40 to 1.62; p = 0.5530 and absolute difference 0.4%). Of the 145 pre-existing PUs of category 2, 89 (61.4%) healed – there was insufficient evidence of a difference in time to healing (Fine and Gray model HR 1.12, 95% CI 0.74 to 1.68; p = 0.6122 and absolute difference 2.9%). Health economics – the within-trial and long-term analysis showed APM to be cost-effective compared with HSFM; however, the difference in costs models are small and the quality-adjusted life-year gains are very small. There were no safety concerns. Blinded photography substudy – the reliability of central blinded review compared with clinical assessment for PUs of category ≥ 2 was ‘very good’ (kappa statistic 0.82, prevalence- and bias-adjusted kappa 0.82). Quality-of-life substudy – the Pressure Ulcer Quality of Life – Prevention (PU-QoL-P) instrument meets the established criteria for reliability, construct validity and responsiveness. Limitations: A lower than anticipated event rate. Conclusions: In acutely ill inpatients who are bedfast/chairfast and/or have a category 1 PU and/or localised skin pain, APMs confer a small treatment phase benefit that is diminished over time. Overall, the APM patient compliance, very low PU incidence rate observed and small differences between mattresses indicate the need for improved indicators for targeting of APMs and individualised decision-making. Decisions should take into account skin status, patient preferences (movement ability and rehabilitation needs) and the presence of factors that may be potentially modifiable through APM allocation, including being completely immobile, having nutritional deficits, lacking capacity and/or having altered skin/category 1 PU. Future work: Explore the relationship between mental capacity, levels of independent movement, repositioning and PU development. Explore ‘what works for whom and in what circumstances’. Trial registration: Current Controlled Trials ISRCTN01151335. Funding: This project was funded by the National Institute for Health Research Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 23, No. 52. See the NIHR Journals Library website for further project information

Researching Zika in pregnancy:lessons for global preparedness

Our understanding of congenital infections is based on prospective studies of women infected during pregnancy. The EU has funded three consortia to study Zika virus, each including a prospective study of pregnant women. Another multi-centre study has been funded by the US National Institutes of Health. This Personal View describes the study designs required to research Zika virus, and questions whether funding academics in the EU and USA to work with collaborators in outbreak areas is an effective strategy. 3 years after the 2015\u201316 Zika virus outbreaks, these collaborations have taught us little about vertical transmission of the virus. In the time taken to approve funding, agree contracts, secure ethics approval, and equip laboratories, Zika virus had largely disappeared. By contrast, prospective studies based on local surveillance and standard-of-care protocols have already provided valuable data. Threats to fetal and child health pose new challenges for global preparedness requiring support for the design and implementation of locally appropriate protocols. These protocols can answer the key questions earlier than externally designed studies and at lower cost. Local protocols can also provide a framework for recruitment of unexposed controls that are required to study less specific outcomes. Other priorities include accelerated development of non-invasive tests, and longer-term storage of neonatal and antenatal samples to facilitate retrospective reconstruction of cohort studies

Vision Screening in Children Aged 6 Months to 5 Years: Evidence Report and Systematic Review for the US Preventive Services Task Force

Importance: Preschool vision screening could allow detection and treatment of vision abnormalities during a critical developmental stage, preserving function and quality of life. Objective: To review the evidence on screening for and treatment of amblyopia, its risk factors, and refractive error in children aged 6 months to 5 years to inform the US Preventive Services Task Force. Data Sources: MEDLINE, Cochrane Library, CINAHL, and trial registries through June 2016; references; and experts, with surveillance of the literature through June 7, 2017. Study Selection: English-language randomized clinical trials (RCTs) or prospective cohort studies that evaluated screening, studies evaluating test accuracy, RCTs of treatment vs inactive controls, and cohort studies or case-control studies assessing harms. Data Extraction and Synthesis: Dual review of abstracts, full-text articles, and study quality; qualitative synthesis of findings. Studies were not quantitatively pooled because of clinical and methodological heterogeneity. Main Outcomes and Measures: Visual acuity, amblyopia, school performance, functioning, quality of life, test accuracy, testability, and harms. Results: Forty studies were included (N = 34 709); 34 evaluated test accuracy. No RCTs compared screening with no screening, and no studies evaluated school performance, function, or quality of life. Studies directly assessing earlier or more intensive screening were limited by high attrition. Positive likelihood ratios were between 5 and 10 for amblyopia risk factors or nonamblyogenic refractive error in most studies of test accuracy and were greater than 10 in most studies evaluating combinations of clinical tests. Inability to cooperate may limit use of some tests in children younger than 3 years. Studies with low prevalence (75%). Among children with amblyopia risk factors (eg, strabismus or anisometropia), patching improved visual acuity of the amblyopic eye by a mean of less than 1 line on a standard chart after 5 to 12 weeks for children pretreated with glasses (2 RCTs, 240 participants); more children treated with patching than with no patching experienced improvement of at least 2 lines (45% vs 21%; P = .003; 1 RCT, 180 participants). Patching plus glasses improved visual acuity by about 1 line after 1 year (0.11 logMAR [95% CI, 0.05-0.17]) for children not pretreated with glasses (1 RCT, 177 participants). Glasses alone improved visual acuity by less than 1 line after 1 year (0.08 logMAR [95% CI, 0.02-0.15], 1 RCT, 177 participants). Conclusions and Relevance: Studies directly evaluating the effectiveness of screening were limited and do not establish whether vision screening in preschool children is better than no screening. Indirect evidence supports the utility of multiple screening tests for identifying preschool children at higher risk for vision problems and the effectiveness of some treatments for improving visual acuity outcomes

Computer-based tools for assessing micro-longitudinal patterns of cognitive function in older adults

Patterns of cognitive change over micro-longitudinal timescales (i.e., ranging from hours to days) are associated with a wide range of age-related health and functional outcomes. However, practical issues with conducting high-frequency assessments make investigations of micro-longitudinal cognition costly and burdensome to run. One way of addressing this is to develop cognitive assessments that can be performed by older adults, in their own homes, without a researcher being present. Here, we address the question of whether reliable and valid cognitive data can be collected over micro-longitudinal timescales using unsupervised cognitive tests.In study 1, 48 older adults completed two touchscreen cognitive tests, on three occasions, in controlled conditions, alongside a battery of standard tests of cognitive functions. In study 2, 40 older adults completed the same two computerized tasks on multiple occasions, over three separate week-long periods, in their own homes, without a researcher present. Here, the tasks were incorporated into a wider touchscreen system (Novel Assessment of Nutrition and Ageing (NANA)) developed to assess multiple domains of health and behavior. Standard tests of cognitive function were also administered prior to participants using the NANA system.Performance on the two “NANA” cognitive tasks showed convergent validity with, and similar levels of reliability to, the standard cognitive battery in both studies. Completion and accuracy rates were also very high. These results show that reliable and valid cognitive data can be collected from older adults using unsupervised computerized tests, thus affording new opportunities for the investigation of cognitive function