High-throughput spheroid screens using volume, resazurin reduction and acid phosphatase activity

Mainstream adoption of physiologically-relevant three-dimensional models has been slow in the last 50 years due to long, manual protocols with poor reproducibility, high price and closed commercial platforms. This chapter describes high-throughput, low-cost, open methods for spheroid viability assessment which use readily-available reagents and open-source software to analyse spheroid volume, metabolism and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination - for both single images and for images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyse plate uniformity (such as edge effects and systematic seeding errors), detect outliers and calculate dose-response. The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time

Spheroid arrays for high-throughput single-cell analysis of spatial patterns and biomarker expression in 3D

We describe and share a device, methodology and image analysis algorithms, which allow up to 66 spheroids to be arranged into a gel-based array directly from a culture plate for downstream processing and analysis. Compared to processing individual samples, the technique uses 11-fold less reagents, saves time and enables automated imaging. To illustrate the power of the technology, we showcase applications of the methodology for investigating 3D spheroid morphology and marker expression and for in vitro safety and efficacy screens. Firstly, spheroid arrays of 11 cell-lines were rapidly assessed for differences in spheroid morphology. Secondly, highly-positive (SOX-2), moderately-positive (Ki-67) and weakly-positive (βIII-tubulin) protein targets were detected and quantified. Third, the arrays enabled screening of ten media compositions for inducing differentiation in human neurospheres. Lastly, the application of spheroid microarrays for spheroid-based drug-screens was demonstrated by quantifying the dose-dependent drop in proliferation and increase in differentiation in etoposide-treated neurospheres

Evidence for the classical integrability of the complete AdS(4) x CP(3) superstring

We construct a zero-curvature Lax connection in a sub-sector of the superstring theory on AdS(4) x CP(3) which is not described by the OSp(6|4)/U(3) x SO(1,3) supercoset sigma-model. In this sub-sector worldsheet fermions associated to eight broken supersymmetries of the type IIA background are physical fields. As such, the prescription for the construction of the Lax connection based on the Z_4-automorphism of the isometry superalgebra OSp(6|4) does not do the job. So, to construct the Lax connection we have used an alternative method which nevertheless relies on the isometry of the target superspace and kappa-symmetry of the Green-Schwarz superstring.Comment: 1+26 pages; v2: minor typos corrected, acknowledgements adde

Transfer RNA-derived small RNAs in the cancer transcriptome

The cellular lifetime includes stages such as differentiation, proliferation, division, senescence and apoptosis.These stages are driven by a strictly ordered process of transcription dynamics. Molecular disruption to RNA polymerase assembly, chromatin remodelling and transcription factor binding through to RNA editing, splicing, post-transcriptional regulation and ribosome scanning can result in significant costs arising from genome instability. Cancer development is one example of when such disruption takes place. RNA silencing is a term used to describe the effects of post-transcriptional gene silencing mediated by a diverse set of small RNA molecules. Small RNAs are crucial for regulating gene expression and microguarding genome integrity.RNA silencing studies predominantly focus on small RNAs such as microRNAs, short-interfering RNAs and piwi-interacting RNAs. We describe an emerging renewal of inter-est in a‘larger’small RNA, the transfer RNA (tRNA).Precisely generated tRNA-derived small RNAs, named tRNA halves (tiRNAs) and tRNA fragments (tRFs), have been reported to be abundant with dysregulation associated with cancer. Transfection of tiRNAs inhibits protein translation by displacing eukaryotic initiation factors from messenger RNA (mRNA) and inaugurating stress granule formation.Knockdown of an overexpressed tRF inhibits cancer cell proliferation. Recovery of lacking tRFs prevents cancer metastasis. The dual oncogenic and tumour-suppressive role is typical of functional small RNAs. We review recent reports on tiRNA and tRF discovery and biogenesis, identification and analysis from next-generation sequencing data and a mechanistic animal study to demonstrate their physiological role in cancer biology. We propose tRNA-derived small RNA-mediated RNA silencing is an innate defence mechanism to prevent oncogenic translation. We expect that cancer cells are percipient to their ablated control of transcription and attempt to prevent loss of genome control through RNA silencing

Success Factors of European Syndromic Surveillance Systems: A Worked Example of Applying Qualitative Comparative Analysis

Introduction: Syndromic surveillance aims at augmenting traditional public health surveillance with timely information. To gain a head start, it mainly analyses existing data such as from web searches or patient records. Despite the setup of many syndromic surveillance systems, there is still much doubt about the benefit of the approach. There are diverse interactions between performance indicators such as timeliness and various system characteristics. This makes the performance assessment of syndromic surveillance systems a complex endeavour. We assessed if the comparison of several syndromic surveillance systems through Qualitative Comparative Analysis helps to evaluate performance and identify key success factors. Materials and Methods: We compiled case-based, mixed data on performance and characteristics of 19 syndromic surveillance systems in Europe from scientific and grey literature and from site visits. We identified success factors by applying crisp-set Qualitative Comparative Analysis. We focused on two main areas of syndromic surveillance application: seasonal influenza surveillance and situational awareness during different types of potentially health threatening events. Results: We found that syndromic surveillance systems might detect the onset or peak of seasonal influenza earlier if they analyse non-clinical data sources. Timely situational awareness during different types of events is supported by an automated syndromic surveillance system capable of analysing multiple syndromes. To our surprise, the analysis of multiple data sources was no key success factor for situational awareness. Conclusions: We suggest to consider these key success factors when designing or further developing syndromic surveillance systems. Qualitative Comparative Analysis helped interpreting complex, mixed data on small-N cases and resulted in concrete and practically relevant findings

Intervention in gene regulatory networks via greedy control policies based on long-run behavior

<p>Abstract</p> <p>Background</p> <p>A salient purpose for studying gene regulatory networks is to derive intervention strategies, the goals being to identify potential drug targets and design gene-based therapeutic intervention. Optimal stochastic control based on the transition probability matrix of the underlying Markov chain has been studied extensively for probabilistic Boolean networks. Optimization is based on minimization of a cost function and a key goal of control is to reduce the steady-state probability mass of undesirable network states. Owing to computational complexity, it is difficult to apply optimal control for large networks.</p> <p>Results</p> <p>In this paper, we propose three new greedy stationary control policies by directly investigating the effects on the network long-run behavior. Similar to the recently proposed mean-first-passage-time (MFPT) control policy, these policies do not depend on minimization of a cost function and avoid the computational burden of dynamic programming. They can be used to design stationary control policies that avoid the need for a user-defined cost function because they are based directly on long-run network behavior; they can be used as an alternative to dynamic programming algorithms when the latter are computationally prohibitive; and they can be used to predict the best control gene with reduced computational complexity, even when one is employing dynamic programming to derive the final control policy. We compare the performance of these three greedy control policies and the MFPT policy using randomly generated probabilistic Boolean networks and give a preliminary example for intervening in a mammalian cell cycle network.</p> <p>Conclusion</p> <p>The newly proposed control policies have better performance in general than the MFPT policy and, as indicated by the results on the mammalian cell cycle network, they can potentially serve as future gene therapeutic intervention strategies.</p

Evaluating the use of the Child and Adolescent Intellectual Disability Screening Questionnaire (CAIDS-Q) to estimate IQ in children with low intellectual ability

In situations where completing a full intellectual assessment is not possible or desirable the clinician or researcher may require an alternative means of accurately estimating intellectual functioning. There has been limited research in the use of proxy IQ measures in children with an intellectual disability or low IQ. The present study aimed to provide a means of converting total scores from a screening tool (the Child and Adolescent Intellectual Disability Screening Questionnaire: CAIDS-Q) to an estimated IQ. A series of linear regression analyses were conducted on data from 428 children and young people referred to clinical services, where FSIQ was predicted from CAIDS-Q total scores. Analyses were conducted for three age groups between ages 6 and 18 years. The study presents a conversion table for converting CAIDS-Q total scores to estimates of FSIQ, with corresponding 95% prediction intervals to allow the clinician or researcher to estimate FSIQ scores from CAIDS-Q total scores. It is emphasised that, while this conversion may offer a quick means of estimating intellectual functioning in children with a below average IQ, it should be used with caution, especially in children aged between 6 and 8 years old

Recoding of Translation in Turtle Mitochondrial Genomes: Programmed Frameshift Mutations and Evidence of a Modified Genetic Code

A +1 frameshift insertion has been documented in the mitochondrial gene nad3 in some birds and reptiles. By sequencing polyadenylated mRNA of the chicken (Gallus gallus), we have shown that the extra nucleotide is transcribed and is present in mature mRNA. Evidence from other animal mitochondrial genomes has led us to hypothesize that certain mitochondrial translation systems have the ability to tolerate frameshift insertions using programmed translational frameshifting. To investigate this, we sequenced the mitochondrial genome of the red-eared slider turtle (Trachemys scripta), where both the widespread nad3 frameshift insertion and a novel site in nad4l were found. Sequencing the region surrounding the insertion in nad3 in a number of other turtles and tortoises reveal general mitochondrial +1 programmed frameshift site features as well as the apparent redefinition of a stop codon in Parker’s snake-neck turtle (Chelodina parkeri), the first known example of this in vertebrate mitochondria

Primordial Black Holes: sirens of the early Universe

Primordial Black Holes (PBHs) are, typically light, black holes which can form in the early Universe. There are a number of formation mechanisms, including the collapse of large density perturbations, cosmic string loops and bubble collisions. The number of PBHs formed is tightly constrained by the consequences of their evaporation and their lensing and dynamical effects. Therefore PBHs are a powerful probe of the physics of the early Universe, in particular models of inflation. They are also a potential cold dark matter candidate.Comment: 21 pages. To be published in "Quantum Aspects of Black Holes", ed. X. Calmet (Springer, 2014

Preoperative calculation of risk for prolonged intensive care unit stay following coronary artery bypass grafting

OBJECTIVE: Patients who have prolonged stay in intensive care unit (ICU) are associated with adverse outcomes. Such patients have cost implications and can lead to shortage of ICU beds. We aimed to develop a preoperative risk prediction tool for prolonged ICU stay following coronary artery surgery (CABG). METHODS: 5,186 patients who underwent CABG between 1st April 1997 and 31st March 2002 were analysed in a development dataset. Logistic regression was used with forward stepwise technique to identify preoperative risk factors for prolonged ICU stay; defined as patients staying longer than 3 days on ICU. Variables examined included presentation history, co-morbidities, catheter and demographic details. The use of cardiopulmonary bypass (CPB) was also recorded. The prediction tool was tested on validation dataset (1197 CABG patients between 1(st )April 2003 and 31(st )March 2004). The area under the receiver operating characteristic (ROC) curve was calculated to assess the performance of the prediction tool. RESULTS: 475(9.2%) patients had a prolonged ICU stay in the development dataset. Variables identified as risk factors for a prolonged ICU stay included renal dysfunction, unstable angina, poor ejection fraction, peripheral vascular disease, obesity, increasing age, smoking, diabetes, priority, hypercholesterolaemia, hypertension, and use of CPB. In the validation dataset, 8.1% patients had a prolonged ICU stay compared to 8.7% expected. The ROC curve for the development and validation datasets was 0.72 and 0.74 respectively. CONCLUSION: A prediction tool has been developed which is reliable and valid. The tool is being piloted at our institution to aid resource management