Combining transcriptional datasets using the generalized singular value decomposition

Background Both microarrays and quantitative real-time PCR are convenient tools for studying the transcriptional levels of genes. The former is preferable for large scale studies while the latter is a more targeted technique. Because of platform-dependent systematic effects, simple comparisons or merging of datasets obtained by these technologies are difficult, even though they may often be desirable. These difficulties are exacerbated if there is only partial overlap between the experimental conditions and genes probed in the two datasets. Results We show here that the generalized singular value decomposition provides a practical tool for merging a small, targeted dataset obtained by quantitative real-time PCR of specific genes with a much larger microarray dataset. The technique permits, for the first time, the identification of genes present in only one dataset co-expressed with a target gene present exclusively in the other dataset, even when experimental conditions for the two datasets are not identical. With the rapidly increasing number of publically available large scale microarray datasets the latter is frequently the case. The method enables us to discover putative candidate genes involved in the biosynthesis of the (1,3;1,4)-β-D-glucan polysaccharide found in plant cell walls. Conclusion We show that the generalized singular value decomposition provides a viable tool for a combined analysis of two gene expression datasets with only partial overlap of both gene sets and experimental conditions. We illustrate how the decomposition can be optimized self-consistently by using a judicious choice of genes to define it. The ability of the technique to seamlessly define a concept of "co-expression" across both datasets provides an avenue for meaningful data integration. We believe that it will prove to be particularly useful for exploiting large, publicly available, microarray datasets for species with unsequenced genomes by complementing them with more limited in-house expression measurements.Andreas W Schreiber, Neil J Shirley, Rachel A Burton and Geoffrey B Finche

Protein Kinase A Activity and Anchoring Are Required for Ovarian Cancer Cell Migration and Invasion

Epithelial ovarian cancer (EOC) is the deadliest of the gynecological malignancies, due in part to its clinically occult metastasis. Therefore, understanding the mechanisms governing EOC dissemination and invasion may provide new targets for antimetastatic therapies or new methods for detection of metastatic disease. The cAMP-dependent protein kinase (PKA) is often dysregulated in EOC. Furthermore, PKA activity and subcellular localization by A-kinase anchoring proteins (AKAPs) are important regulators of cytoskeletal dynamics and cell migration. Thus, we sought to study the role of PKA and AKAP function in both EOC cell migration and invasion. Using the plasma membrane-directed PKA biosensor, pmAKAR3, and an improved migration/invasion assay, we show that PKA is activated at the leading edge of migrating SKOV-3 EOC cells, and that inhibition of PKA activity blocks SKOV-3 cell migration. Furthermore, we show that while the PKA activity within the leading edge of these cells is mediated by anchoring of type-II regulatory PKA subunits (RII), inhibition of anchoring of either RI or RII PKA subunits blocks cell migration. Importantly, we also show – for the first time – that PKA activity is up-regulated at the leading edge of SKOV-3 cells during invasion of a three-dimensional extracellular matrix and, as seen for migration, inhibition of either PKA activity or AKAP-mediated PKA anchoring blocks matrix invasion. These data are the first to demonstrate that the invasion of extracellular matrix by cancer cells elicits activation of PKA within the invasive leading edge and that both PKA activity and anchoring are required for matrix invasion. These observations suggest a role for PKA and AKAP activity in EOC metastasis

The dynamics of transcript abundance during cellularization of developing barley endosperm

Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development.Runxuan Zhang, Matthew R. Tucker, Rachel A Burton, Neil J. Shirley, Alan Little, Jenny Morris, Linda Milne, Kelly Houston, Pete E. Hedley, Robbie Waugh, and Geoffrey B. Finche

Evaluating the End-User Experience of Private Browsing Mode

Nowadays, all major web browsers have a private browsing mode. However, the mode's benefits and limitations are not particularly understood. Through the use of survey studies, prior work has found that most users are either unaware of private browsing or do not use it. Further, those who do use private browsing generally have misconceptions about what protection it provides. However, prior work has not investigated \emph{why} users misunderstand the benefits and limitations of private browsing. In this work, we do so by designing and conducting a three-part study: (1) an analytical approach combining cognitive walkthrough and heuristic evaluation to inspect the user interface of private mode in different browsers; (2) a qualitative, interview-based study to explore users' mental models of private browsing and its security goals; (3) a participatory design study to investigate why existing browser disclosures, the in-browser explanations of private browsing mode, do not communicate the security goals of private browsing to users. Participants critiqued the browser disclosures of three web browsers: Brave, Firefox, and Google Chrome, and then designed new ones. We find that the user interface of private mode in different web browsers violates several well-established design guidelines and heuristics. Further, most participants had incorrect mental models of private browsing, influencing their understanding and usage of private mode. Additionally, we find that existing browser disclosures are not only vague, but also misleading. None of the three studied browser disclosures communicates or explains the primary security goal of private browsing. Drawing from the results of our user study, we extract a set of design recommendations that we encourage browser designers to validate, in order to design more effective and informative browser disclosures related to private mode

Physiological and behavioral effects of animal-assisted interventions for therapy dogs in pediatric oncology settings

Over the past two decades, animal-assisted interventions (AAIs), defined as the purposeful incorporation of specially trained animals in services to improve human health, have become increasingly popular in clinical settings. However, to date, there have been few rigorously-designed studies aimed at examining the impact of AAIs on therapy animals, despite a notable potential for stress. The current study measured physiological and behavioral stress indicators in therapy dogs who participated in AAI sessions in pediatric oncology settings, while also examining the psychosocial effects for patients and their parents. This manuscript describes the study’s canine stress findings. Methods: A total of 26 therapy dog-handler teams were paired with newly diagnosed children with cancer at five children’s hospitals in the United States. These teams provided regular AAI visits to the child and his/her parent(s) for a period of four months. The teams completed a demographic form, the Canine Behavioral Assessment & Research Questionnaire (C-BARQ), and a self-report survey to document the types of activities that occurred during each session. Canine saliva was also collected at five baseline time points and 20 minutes after the start of study sessions for cortisol analysis, and all study sessions were video recorded to document the dog’s behavior via an ethogram measure. Results: Data showed no significant differences in salivary cortisol levels between baseline (0.51µg/dL) and AAI sessions (0.44µg/dL), p = 0.757. Higher salivary cortisol was significantly associated with a higher number of stress behaviors per session (p = 0.039). There was a significant relationship between stress and affiliative session behaviors (pConclusions:Results show that therapy dogs did not have significantly increased physiological stress responses, nor did they exhibit significantly more stress-related behaviors than affiliative-related behaviors, while participating in AAIs in pediatric oncology settings. The significant relationship between canine cortisol and behavior, thus strengthening the argument for the use of cortisol in canine well-being research. This study discusses the importance of further investigation to confirm these findings, which may lead to enhanced canine involvement in hospital settings

The bulk mineralogy, elemental composition, and water content of the Winchcombe CM chondrite fall

On the micro-scale, the Winchcombe CM carbonaceous chondrite contains a number of lithological units with a variety of degrees of aqueous alteration. However, an understanding of the average hydration state is useful when comparing to other meteorites and remote observations of airless bodies. We report correlated bulk analyses on multiple subsamples of the Winchcombe meteorite, determining an average phyllosilicate fraction (PSF) petrologic type of 1.2 and an average water content of 11.9 wt%. We show the elemental composition and distribution of iron and iron oxidation state are consistent with measurements from other CM chondrites, however Winchcombe shows a low Hg concentration of 58.1 ±0.5 ng/g. We demonstrate that infrared reflectance spectra of Winchcombe are consistent with its bulk modal mineralogy, and comparable to other CM chondrites with similar average petrologic types. Finally, we also evaluate whether spectral parameters can estimate H/Si ratios and water abundances, finding generally spectral parameters underestimate water abundance compared to measured values.Output Status: Forthcoming/Available Onlin

Transcript Profiling of MIKCc MADS-Box Genes Reveals Conserved and Novel Roles in Barley Inflorescence Development

MADS-box genes have a wide range of functions in plant reproductive development and grain production. The ABCDE model of floral organ development shows that MADS-box genes are central players in these events in dicotyledonous plants but the applicability of this model remains largely unknown in many grass crops. Here, we show that transcript analysis of all MIKCc MADS-box genes through barley (Hordeum vulgare L.) inflorescence development reveals co-expression groups that can be linked to developmental events. Thirty-four MIKCc MADS-box genes were identified in the barley genome and single-nucleotide polymorphism (SNP) scanning of 22,626 barley varieties revealed that the natural variation in the coding regions of these genes is low and the sequences have been extremely conserved during barley domestication. More detailed transcript analysis showed that MADS-box genes are generally expressed at key inflorescence developmental phases and across various floral organs in barley, as predicted by the ABCDE model. However, expression patterns of some MADS genes, for example HvMADS58 (AGAMOUS subfamily) and HvMADS34 (SEPALLATA subfamily), clearly deviate from predicted patterns. This places them outside the scope of the classical ABCDE model of floral development and demonstrates that the central tenet of antagonism between A- and C-class gene expression in the ABC model of other plants does not occur in barley. Co-expression across three correlation sets showed that specifically grouped members of the barley MIKCc MADS-box genes are likely to be involved in developmental events driving inflorescence meristem initiation, floral meristem identity and floral organ determination. Based on these observations, we propose a potential floral ABCDE working model in barley, where the classic model is generally upheld, but that also provides new insights into the role of MIKCc MADS-box genes in the developing barley inflorescence.Hendrik N. J. Kuijer, Neil J. Shirley, Shi F. Khor, Jin Shi, Julian Schwerdt, Dabing Zhang, Gang Li, and Rachel A. Burto

Evolution of multiple cell clones over a 29-year period of a CLL patient

Chronic lymphocytic leukaemia (CLL) is a frequent B-cell malignancy, characterized by recurrent somatic chromosome alterations and a low level of point mutations. Here we present single-nucleotide polymorphism microarray analyses of a single CLL patient over 29 years of observation and treatment, and transcriptome and whole-genome sequencing at selected time points. We identify chromosome alterations 13q14−, 6q− and 12q+ in early cell clones, elimination of clonal populations following therapy, and subsequent appearance of a clone containing trisomy 12 and chromosome 10 copy-neutral loss of heterogeneity that marks a major population dominant at death. Serial single-cell RNA sequencing reveals an expression pattern with high FOS, JUN and KLF4 at disease acceleration, which resolves following therapy, but reoccurs following relapse and death. Transcriptome evolution indicates complex changes in expression occur over time. In conclusion, CLL can evolve gradually during indolent phases, and undergo rapid changes following therapy

Loss of Expression and Promoter Methylation of SLIT2 Are Associated with Sessile Serrated Adenoma Formation.

Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing) and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1–4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson “two hit” hypothesis