Cell type-specific profiling of protein-DNA interactions without cell isolation using Targeted DamID with next-generation sequencing

The ability to profile transcription and chromatin binding in a cell type-specific manner is a powerful approach for understanding cell fate specification and cellular function in multicellular organisms. We recently developed Targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a Protocol Extension, this article describes substantial modifications to an existing Protocol and offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, avoiding toxicity and potential artefacts from over-expression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to Nextgeneration Sequencing technology. TaDa is robust, reproducible, and highly sensitive. Compared to other methods for cell-type specific profiling, the technique requires no cell-sorting, crosslinking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II, TaDa can also identify transcribed genes in a cell type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure – from collecting tissue samples to generating sequencing libraries – can be accomplished within 5 days

Return to driving after traumatic brain injury : a British perspective

Primary Objective: to identify current legal situation, and professional practice in assisting persons with traumatic brain injury (TBI) to return to safe driving after injury. Methods and Procedures A brief review of relevant literature, a description of the current statutory and quasi-statutory authorities regulating return to driving after TBI in the UK, and a description of the nature and resolution of clinical and practical dilemmas facing professionals helping return to safe driving after TBI. Each of the 15 UK mobility centres was contacted and literature requested; in addition a representative of each centre responded to a structured telephone survey. Main Outcome and Results: The current situation in Great Britain is described, with a brief analysis of the strengths and weaknesses both of the current statutory situation, and also the practical situation (driving centres), with suggestions for improvements in practice. Conclusion Although brain injury may cause serious limitations in driving ability, previous drivers are not routinely assessed or advised regarding return to driving after TBI

Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1.

The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified 'β-clasp' structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets

Absence of a Spin Liquid Phase in the Hubbard Model on the Honeycomb Lattice

A spin liquid is a novel quantum state of matter with no conventional order parameter where a finite charge gap exists even though the band theory would predict metallic behavior. Finding a stable spin liquid in two or higher spatial dimensions is one of the most challenging and debated issues in condensed matter physics. Very recently, it has been reported that a model of graphene, i.e., the Hubbard model on the honeycomb lattice, can show a spin liquid ground state in a wide region of the phase diagram, between a semi-metal (SM) and an antiferromagnetic insulator (AFMI). Here, by performing numerically exact quantum Monte Carlo simulations, we extend the previous study to much larger clusters (containing up to 2592 sites), and find, if any, a very weak evidence of this spin liquid region. Instead, our calculations strongly indicate a direct and continuous quantum phase transition between SM and AFMI.Comment: 15 pages with 7 figures and 9 tables including supplementary information, accepted for publication in Scientific Report

Comparison of techniques for handling missing covariate data within prognostic modelling studies: a simulation study

Background: There is no consensus on the most appropriate approach to handle missing covariate data within prognostic modelling studies. Therefore a simulation study was performed to assess the effects of different missing data techniques on the performance of a prognostic model. Methods: Datasets were generated to resemble the skewed distributions seen in a motivating breast cancer example. Multivariate missing data were imposed on four covariates using four different mechanisms; missing completely at random (MCAR), missing at random (MAR), missing not at random (MNAR) and a combination of all three mechanisms. Five amounts of incomplete cases from 5% to 75% were considered. Complete case analysis (CC), single imputation (SI) and five multiple imputation (MI) techniques available within the R statistical software were investigated: a) data augmentation (DA) approach assuming a multivariate normal distribution, b) DA assuming a general location model, c) regression switching imputation, d) regression switching with predictive mean matching (MICE-PMM) and e) flexible additive imputation models. A Cox proportional hazards model was fitted and appropriate estimates for the regression coefficients and model performance measures were obtained. Results: Performing a CC analysis produced unbiased regression estimates, but inflated standard errors, which affected the significance of the covariates in the model with 25% or more missingness. Using SI, underestimated the variability; resulting in poor coverage even with 10% missingness. Of the MI approaches, applying MICE-PMM produced, in general, the least biased estimates and better coverage for the incomplete covariates and better model performance for all mechanisms. However, this MI approach still produced biased regression coefficient estimates for the incomplete skewed continuous covariates when 50% or more cases had missing data imposed with a MCAR, MAR or combined mechanism. When the missingness depended on the incomplete covariates, i.e. MNAR, estimates were biased with more than 10% incomplete cases for all MI approaches. Conclusion: The results from this simulation study suggest that performing MICE-PMM may be the preferred MI approach provided that less than 50% of the cases have missing data and the missing data are not MNAR

Achieving sustainable quality in maternity services – using audit of incontinence and dyspareunia to identify shortfalls in meeting standards

BACKGROUND: Some complications of childbirth (for example, faecal incontinence) are a source of social embarrassment for women, and are often under reported. Therefore, it was felt important to determine levels of complications (against established standards) and to consider obstetric measures aimed at reducing them. METHODS: Clinical information was collected on 1036 primiparous women delivering at North and South Staffordshire Acute and Community Trusts over a 5-month period in 1997. A questionnaire was sent to 970 women which included self-assessment of levels of incontinence and dyspareunia prior to pregnancy, at 6 weeks post delivery and 9 to 14 months post delivery. RESULTS: The response rate was 48%(470/970). Relatively high levels of obstetric interventions were found. In addition, the rates of instrumental deliveries differed between the two hospitals. The highest rates of postnatal symptoms had occurred at 6 weeks, but for many women problems were still present at the time of the survey. At 9–14 months high rates of dyspareunia (29%(102/347)) and urinary incontinence (35%(133/382)) were reported. Seventeen women (4%) complained of faecal incontinence at this time. Similar rates of urinary incontinence and dyspareunia were seen regardless of mode of delivery. CONCLUSION: Further work should be undertaken to reduce the obstetric interventions, especially instrumental deliveries. Improvements in a number of areas of care should be undertaken, including improved patient information, improved professional communication and improved professional recognition and management of third degree tears. It is likely that these measures would lead to a reduction in incontinence and dyspareunia after childbirth

Poleward energy transport: is the standard definition physically relevant at all time scales?

Poleward energy transport in the atmosphere and oceans constitutes an important branch of the global energy budget, and its role in the climate system has been the subject of many studies. In the atmosphere, the transport is affected by “eddies” and large scale meridional cells, both with zero net mass transport across latitude circles, but also partly by processes associated with a net transport of mass across latitude circles. The latter must cease to operate in steady state, but they may be significant when time variability of the heat budget is considered. Indeed, examination of reanalysis data on short (daily to monthly) timescales shows that mass variations on these timescales result in surprisingly large fluctuations (in excess of 10^15 W = 1 PW) in the poleward heat transport. These fluctuations are referred to as “extensive”, for they primarily alter the mass integrated energy of the region considered, but not its averaged value. It is suggested that extensive fluctuations mask more meaningful climate signals present in the heat transport variability on monthly and interannual timescales, and a new formulation is proposed to isolate the latter. This new formulation is applied successfully to reanalysis data and climate model simulations

Microwave amplification with nanomechanical resonators

Sensitive measurement of electrical signals is at the heart of modern science and technology. According to quantum mechanics, any detector or amplifier is required to add a certain amount of noise to the signal, equaling at best the energy of quantum fluctuations. The quantum limit of added noise has nearly been reached with superconducting devices which take advantage of nonlinearities in Josephson junctions. Here, we introduce a new paradigm of amplification of microwave signals with the help of a mechanical oscillator. By relying on the radiation pressure force on a nanomechanical resonator, we provide an experimental demonstration and an analytical description of how the injection of microwaves induces coherent stimulated emission and signal amplification. This scheme, based on two linear oscillators, has the advantage of being conceptually and practically simpler than the Josephson junction devices, and, at the same time, has a high potential to reach quantum limited operation. With a measured signal amplification of 25 decibels and the addition of 20 quanta of noise, we anticipate near quantum-limited mechanical microwave amplification is feasible in various applications involving integrated electrical circuits.Comment: Main text + supplementary information. 14 pages, 3 figures (main text), 18 pages, 6 figures (supplementary information