Thousands Of Conditions Research Articles

Cell cycle visualization tools to study cardiomyocyte proliferation in real-time.

Cardiomyocytes in the adult human heart are quiescent and those lost following heart injury are not replaced by proliferating survivors. Considerable effort has been made to understand the mechanisms underlying cardiomyocyte cell cycle exit and re-entry, with view to discovering therapeutics that could stimulate cardiomyocyte proliferation and heart regeneration. The advent of large compound libraries and robotic liquid handling platforms has enabled the screening of thousands of conditions in a single experiment but success of these screens depends on the appropriateness and quality of the model used. Quantification of (human) cardiomyocyte proliferation in high throughput has remained problematic because conventional antibody-based staining is costly, technically challenging and does not discriminate between cardiomyocyte division and failure in karyokinesis or cytokinesis. Live cell imaging has provided alternatives that facilitate high-throughput screening but these have other limitations. Here, we (i) review the cell cycle features of cardiomyocytes, (ii) discuss various cell cycle fluorescent reporter systems, and (iii) speculate on what could improve their predictive value in the context of cardiomyocyte proliferation. Finally, we consider how these new methods can be used in combination with state-of-the-art three-dimensional human cardiac organoid platforms to identify pro-proliferative signalling pathways that could stimulate regeneration of the human heart.

High-throughput single particle tracking reveals estrogen receptor protein interaction landscape in living cells.

Protein motion is defined by a network of interactions within the cellular environment, serving as a direct report of activity and function. The key challenges in single particle tracking (SPT) studies in live cells remain the assignment of biological meaning from a set of molecule trajectories, as well as the technical challenges in generating the volume of data necessary to characterize interaction pathways. Through advancements in optical engineering, automation, and computation, we have developed an industrial-scale system to track fast-moving proteins across thousands of conditions in mammalian cell lines. We applied this system to study the estrogen receptor (ER), a protein well known for its role in normal human development as well as its prominent role in many breast cancers. We present a chemical genetics screen of thousands of known bioactive compounds to identify novel ER modulators. Intriguingly, chemical genetics also revealed the contributions of multiple pathway interactions on ER dynamics, distinguishable through pathway-specific effects. Further, SPT can be an effective readout for direct target engagement via real-time kinetics measurements, can elucidate structure-activity relationships, and can predict compound efficacy in orthogonal assays. Taken together, our results underscore the wealth of information embedded within high-throughput, live-cell SPT data, and the utility of this information in furthering our understanding of biology across a broad set of applications.

Optimizing Associative Experimental Design for Protein Crystallization Screening.

The goal of protein crystallization screening is the determination of the main factors of importance to crystallizing the protein under investigation. One of the major issues about determining these factors is that screening is often expanded to many hundreds or thousands of conditions to maximize combinatorial chemical space coverage for maximizing the chances of a successful (crystalline) outcome. In this paper, we propose an experimental design method called "Associative Experimental Design (AED)" and an optimization method includes eliminating prohibited combinations and prioritizing reagents based on AED analysis of results from protein crystallization experiments. AED generates candidate cocktails based on these initial screening results. These results are analyzed to determine those screening factors in chemical space that are most likely to lead to higher scoring outcomes, crystals. We have tested AED on three proteins derived from the hyperthermophile Thermococcus thioreducens, and we applied an optimization method to these proteins. Our AED method generated novel cocktails (count provided in parentheses) leading to crystals for three proteins as follows: Nucleoside diphosphate kinase (4), HAD superfamily hydrolase (2), Nucleoside kinase (1). After getting promising results, we have tested our optimization method on four different proteins. The AED method with optimization yielded 4, 3, and 20 crystalline conditions for holo Human Transferrin, archaeal exosome protein, and Nucleoside diphosphate kinase, respectively.

Initial Results Show Medicare Savings From Some ACOs

Fewer hospital readmissions, high-cost imaging studies, and emergency department visits helped 29 of 114 accountable care organizations earn $126 million in shared Medicare savings in 2012, the Centers for Medicare & Medicaid Services announced. CMS issued interim financial results for the first year that ACOs participated in the Medicare Shared Savings Program. Under the program, ACOs that reduce costs while providing high-quality care can share in the savings to the federal program. They must report on 33 quality measures relating to care coordination and patient safety, use of appropriate preventive health services, improved care for at-risk populations, and patient and caregiver experience of care. Of 114 ACOs that started in 2012, 54 spent less than anticipated, and 29 of the 54 collectively received $126 million as their share of the savings. Medicare kept $128 million of savings. “What I think is impressive is that so many of the organizations did save in the first year,” CMS Principal Deputy Administrator Jon Blum said. The agency had not anticipated savings – especially large savings – he said. CMS would not reveal which ACOs met their benchmarks, which were compensated for saving, or how much any ACO received. The agency also announced that 232 acute care hospitals, skilled nursing homes, physician group practices, long-term care hospitals, and home health agencies were moving into the next phase of the Bundled Payments for Care Improvement initiative. The participants were announced in January 2013 and spent the year preparing to be accountable for episodes of care involving some 48 clinical conditions. The goal of the program is to encourage providers to work together to coordinate care for patients while in the hospital and afterward. This year, those 232 providers are bearing risk for the care they provide. The agency has also added thousands of conditions that can become eligible for shared risk, said Patrick Conway, MD, deputy administrator for innovation and quality at the CMS.

A Straightforward and Robust Method for Introducing Human Hair as a Nucleant into High Throughput Crystallization Trials

Growing X-ray grade crystals of a specific protein is a process of trial and error. Usually, hundreds or even thousands of conditions are screened in order to identify useful crystallization conditions. Heterogeneous nucleants have been shown to increase the success rate of crystallization trials, and (human) hair has previously been identified as a promising nucleant. Here, we describe and evaluate a method for preparing crystallization plates that are locally coated with fragments of human hair, allowing automated, high-throughput crystallization trials in a fashion that is entirely compatible with standard hanging or sitting drop crystallization techniques. We assessed the effect of these nucleants on the crystallization of 11 different proteins in more than 4000 crystallization trials. We found additional crystallization conditions for 10 out of 11 proteins when using the standard JCSG+ screen (96 different conditions). In total, 34 additional crystallization conditions could be identified (13.1% of the total number of successful crystallizations). The increase in crystallization conditions ranged between 33.3% (two additional conditions were identified for myoglobin on top of four homogeneous crystallizations) to 1.2% (we identified a single additional condition for insulin, which crystallized in 85 out of 96 conditions); the median increase in crystallization hits was 14%. On the basis of these numbers, we conclude that the inclusion of human hair fragments in high throughput crystallization screens may be beneficial. The method is inexpensive, straightforward with standard equipment and uses materials available in any crystallization lab. Furthermore, initial experiments with the crystallization of membrane proteins on hair show the technique may also be beneficial for growing membrane proteins.

The influence of aqueous content in small scale salt screening—Improving hit rate for weakly basic, low solubility drugs

Salt screening and selection is a well established approach for improving the properties of drug candidates, including dissolution rate and bioavailability. Typically during early development only small amounts of compound are available for solid state profiling, including salt screening. In order to probe large areas of experimental space, high-throughput screening is utilized and is often designed in a way to search for suitable crystallization parameters within hundreds or even thousands of conditions. However, the hit rate in these types of screens can be very low.In order to allow for selection of a salt form early within the drug development process whilst using smaller amounts of compounds, a screening procedure taking into account the compounds properties and the driving forces for salt formation is described. Experiments were carried out on the model compounds clotrimazole, cinnarizine itraconazole and atropine. We found an increase in crystalline hit rate for water-insoluble drugs crystallized from solutions that included at least 10% aqueous content.Conversely it was observed that compounds with greater water solubility did not benefit from aqueous content in salt screening, instead organic solvents lead to more crystalline screening hits. Results from four model compounds show that the inclusion of an aqueous component to the salt reaction can enhance the chance of salt formation and significantly improve the crystalline hit rate for low water soluble drugs.

Martin Brodie: epilepsy expertise and whisky wisdom

Martin Brodie: epilepsy expertise and whisky wisdom

QUBIC: a qualitative biclustering algorithm for analyses of gene expression data

Biclustering extends the traditional clustering techniques by attempting to find (all) subgroups of genes with similar expression patterns under to-be-identified subsets of experimental conditions when applied to gene expression data. Still the real power of this clustering strategy is yet to be fully realized due to the lack of effective and efficient algorithms for reliably solving the general biclustering problem. We report a QUalitative BIClustering algorithm (QUBIC) that can solve the biclustering problem in a more general form, compared to existing algorithms, through employing a combination of qualitative (or semi-quantitative) measures of gene expression data and a combinatorial optimization technique. One key unique feature of the QUBIC algorithm is that it can identify all statistically significant biclusters including biclusters with the so-called ‘scaling patterns’, a problem considered to be rather challenging; another key unique feature is that the algorithm solves such general biclustering problems very efficiently, capable of solving biclustering problems with tens of thousands of genes under up to thousands of conditions in a few minutes of the CPU time on a desktop computer. We have demonstrated a considerably improved biclustering performance by our algorithm compared to the existing algorithms on various benchmark sets and data sets of our own. QUBIC was written in ANSI C and tested using GCC (version 4.1.2) on Linux. Its source code is available at: http://csbl.bmb.uga.edu/∼maqin/bicluster. A server version of QUBIC is also available upon request.

The Importance of Gene—Environment Interaction

Given recent genetic advances, it is not surprising that genetics information is increasingly being used to improve health care. Thousands of conditions caused by single genes (Mendelian diseases) have been identified over the last century. However, Mendelian diseases are rare; thus, few individuals directly benefit from gene identification. In contrast, common complex diseases, such as obesity, breast cancer, and depression, directly affect many more individuals. Common complex diseases are caused by multiple genes, environmental factors, and/or interaction of genetic and environmental factors. This article provides a framework for the successful conduct of gene—environment studies. To accomplish this goal, the basic study designs and procedures of implementation for gene—environment interaction are described. Next, examples of gene—environment interaction in obesity epidemiology are reviewed. Last, the authors review reasons why epidemiological studies that incorporate gene—environment interaction have been unable to demonstrate statistically significant interactions and why conflicting results are reported.

The role of CFD in aerodynamics, off-design

AbstractWe discuss the status, trends and long-term ambitions of Computational Fluid Dynamics (CFD) when applied away from the design point or concept, and therefore in the historically weak areas of CFD. This includes both undesirable flight conditions, such as stall, and undesirable products of the flow, such as noise. All pose the great challenge of turbulence, and accuracy is as dependent on the ideas behind the turbulence treatment as it is on computing power. A measured shift from Reynolds-averaged representations to large-eddy simulations will take place. Empiricism, both turbulence and engineering related, will recede only step by step over many years. Yet, CFD will make full use of every increase in computer power of this century. Increasingly, CFD will compete with flight tests, not just with wind tunnels, and will be validated by flight tests. Integration with other disciplines will allow us to predict crucial phenomena such as flutter, sonic fatigue, and pilot-induced oscillations. A gap will remain at any time between the phenomena amenable to a ‘grand challenge’ calculation and those amenable to a fully CFD-based design process, because certification involves thousands of conditions. The highest demand currently is in community and cabin noise for which industrial-accuracy methods are non-existent. On the other hand, gratifying progress has occurred in the area of stall and spin.