Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput leve

Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein–DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level

Real-Time Position Reconstruction with Hippocampal Place Cells

Brain–computer interfaces (BCI) are using the electroencephalogram, the electrocorticogram and trains of action potentials as inputs to analyze brain activity for communication purposes and/or the control of external devices. Thus far it is not known whether a BCI system can be developed that utilizes the states of brain structures that are situated well below the cortical surface, such as the hippocampus. In order to address this question we used the activity of hippocampal place cells (PCs) to predict the position of an rodent in real-time. First, spike activity was recorded from the hippocampus during foraging and analyzed off-line to optimize the spike sorting and position reconstruction algorithm of rats. Then the spike activity was recorded and analyzed in real-time. The rat was running in a box of 80 cm × 80 cm and its locomotor movement was captured with a video tracking system. Data were acquired to calculate the rat's trajectories and to identify place fields. Then a Bayesian classifier was trained to predict the position of the rat given its neural activity. This information was used in subsequent trials to predict the rat's position in real-time. The real-time experiments were successfully performed and yielded an error between 12.2 and 17.4% using 5–6 neurons. It must be noted here that the encoding step was done with data recorded before the real-time experiment and comparable accuracies between off-line (mean error of 15.9% for three rats) and real-time experiments (mean error of 14.7%) were achieved. The experiment shows proof of principle that position reconstruction can be done in real-time, that PCs were stable and spike sorting was robust enough to generalize from the training run to the real-time reconstruction phase of the experiment. Real-time reconstruction may be used for a variety of purposes, including creating behavioral–neuronal feedback loops or for implementing neuroprosthetic control

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays

Intervenir sobre la cultura organizacional: ¿qué aspectos se pueden considerar?

La cultura organizacional (co) es un macroconstructo que involucra una gran variedad de componentes y funciones organizacionales (Warner, 2014). Reyes y Moros (2018) señalan que tiene su origen en el estudio realizado en Hawthorne por Elton Mayo y otros investigadores de la Escuela de las Relaciones Humanas de la Administración, en el que buscaban identificar la influencia de las condiciones físicas y ambientales en el desempeño individual. Para Reyes y Moros (2018), la co se siguió desarrollando en los años setenta con Pettigrew, para ser entendida como un sistema de significados que tanto pública como colectivamente es aceptado para operar en un tiempo y por un grupo determinado. Los autores la definen como “… un sistema de significados compartidos por los miembros de la organización, los cuales son el resultado de una construcción social constituida a través de símbolos y como tal deben ser interpretados”1a edició

Origin and function of stomata in the moss Physcomitrella patens.

Stomata are microscopic valves on plant surfaces that originated over 400 million years (Myr) ago and facilitated the greening of Earth's continents by permitting efficient shoot-atmosphere gas exchange and plant hydration(1). However, the core genetic machinery regulating stomatal development in non-vascular land plants is poorly understood(2-4) and their function has remained a matter of debate for a century(5). Here, we show that genes encoding the two basic helix-loop-helix proteins PpSMF1 (SPEECH, MUTE and FAMA-like) and PpSCREAM1 (SCRM1) in the moss Physcomitrella patens are orthologous to transcriptional regulators of stomatal development in the flowering plant Arabidopsis thaliana and essential for stomata formation in moss. Targeted P. patens knockout mutants lacking either PpSMF1 or PpSCRM1 develop gametophytes indistinguishable from wild-type plants but mutant sporophytes lack stomata. Protein-protein interaction assays reveal heterodimerization between PpSMF1 and PpSCRM1, which, together with moss-angiosperm gene complementations(6), suggests deep functional conservation of the heterodimeric SMF1 and SCRM1 unit is required to activate transcription for moss stomatal development, as in A. thaliana(7). Moreover, stomata-less sporophytes of ΔPpSMF1 and ΔPpSCRM1 mutants exhibited delayed dehiscence, implying stomata might have promoted dehiscence in the first complex land-plant sporophytes

Remediation programmes for practising doctors to restore patient safety: the RESTORE realist review

Background An underperforming doctor puts patient safety at risk. Remediation is an intervention intended to address underperformance and return a doctor to safe practice. Used in health-care systems all over the world, it has clear implications for both patient safety and doctor retention in the workforce. However, there is limited evidence underpinning remediation programmes, particularly a lack of knowledge as to why and how a remedial intervention may work to change a doctor’s practice. Objectives To (1) conduct a realist review of the literature to ascertain why, how, in what contexts, for whom and to what extent remediation programmes for practising doctors work to restore patient safety; and (2) provide recommendations on tailoring, implementation and design strategies to improve remediation interventions for doctors. Design A realist review of the literature underpinned by the Realist And MEta-narrative Evidence Syntheses: Evolving Standards quality and reporting standards. Data sources Searches of bibliographic databases were conducted in June 2018 using the following databases: EMBASE, MEDLINE, Cumulative Index to Nursing and Allied Health Literature, PsycINFO, Education Resources Information Center, Database of Abstracts of Reviews of Effects, Applied Social Sciences Index and Abstracts, and Health Management Information Consortium. Grey literature searches were conducted in June 2019 using the following: Google Scholar (Google Inc., Mountain View, CA, USA), OpenGrey, NHS England, North Grey Literature Collection, National Institute for Health and Care Excellence Evidence, Electronic Theses Online Service, Health Systems Evidence and Turning Research into Practice. Further relevant studies were identified via backward citation searching, searching the libraries of the core research team and through a stakeholder group. Review methods Realist review is a theory-orientated and explanatory approach to the synthesis of evidence that seeks to develop programme theories about how an intervention produces its effects. We developed a programme theory of remediation by convening a stakeholder group and undertaking a systematic search of the literature. We included all studies in the English language on the remediation of practising doctors, all study designs, all health-care settings and all outcome measures. We extracted relevant sections of text relating to the programme theory. Extracted data were then synthesised using a realist logic of analysis to identify context–mechanism–outcome configurations. Results A total of 141 records were included. Of the 141 studies included in the review, 64% related to North America and 14% were from the UK. The majority of studies (72%) were published between 2008 and 2018. A total of 33% of articles were commentaries, 30% were research papers, 25% were case studies and 12% were other types of articles. Among the research papers, 64% were quantitative, 19% were literature reviews, 14% were qualitative and 3% were mixed methods. A total of 40% of the articles were about junior doctors/residents, 31% were about practicing physicians, 17% were about a mixture of both (with some including medical students) and 12% were not applicable. A total of 40% of studies focused on remediating all areas of clinical practice, including medical knowledge, clinical skills and professionalism. A total of 27% of studies focused on professionalism only, 19% focused on knowledge and/or clinical skills and 14% did not specify. A total of 32% of studies described a remediation intervention, 16% outlined strategies for designing remediation programmes, 11% outlined remediation models and 41% were not applicable. Twenty-nine context–mechanism–outcome configurations were identified. Remediation programmes work when they develop doctors’ insight and motivation, and reinforce behaviour change. Strategies such as providing safe spaces, using advocacy to develop trust in the remediation process and carefully framing feedback create contexts in which psychological safety and professional dissonance lead to the development of insight. Involving the remediating doctor in remediation planning can provide a perceived sense of control in the process and this, alongside correcting causal attribution, goal-setting, destigmatising remediation and clarity of consequences, helps motivate doctors to change. Sustained change may be facilitated by practising new behaviours and skills and through guided reflection. Limitations Limitations were the low quality of included literature and limited number of UK-based studies. Future work Future work should use the recommendations to optimise the delivery of existing remediation programmes for doctors in the NHS. Study registration This study is registered as PROSPERO CRD42018088779. Funding This project was funded by the National Institute for Health Research (NIHR) Health Services and Delivery Research programme and will be published in full in Health Services and Delivery Research; Vol. 9, No. 11. See the NIHR Journals Library website for further project information. </jats:sec