Adaptation of a RAS pathway activation signature from FF to FFPE tissues in colorectal cancer

Paper presented at the 4th Strathmore International Mathematics Conference (SIMC 2017), 19 - 23 June 2017, Strathmore University, Nairobi, Kenya.Background: The KRAS gene is mutated in about 40 % of colorectal cancer (CRC) cases, which has been clinically validated as a predictive mutational marker of intrinsic resistance to anti-EGFR inhibitor (EGFRi) therapy. Since nearly 60 % of patients with a wild type KRAS fail to respond to EGFRicombination therapies, there is a need to develop more reliable molecular signatures to better predict response. Here we address the challenge of adapting a gene expression signature predictive of RAS pathway activation, created using fresh frozen (FF) tissues, for use with more widely available formalinfixed paraffin-embedded (FFPE) tissues. Methods: In this study, we evaluated the translation of an 18-gene RAS pathway signature score from FF to FFPE in 54 CRC cases, using a head-to- head comparison of five technology platforms. FFPE-based technologies included the Affymetrix Gene Chip (Affy), NanoString nCounter™ (NanoS),Illumina whole genome RNASeq (RNA-Acc), Illumina targeted RNASeq (t-RNA), and Illuminastranded Total RNA-rRNA- depletion (rRNA). Results: Using Affy_FF as the “gold” standard, initial analysis of the 18-gene RAS scores on all 54samples shows varying pairwise Spearman correlations, with (1) Affy_FFPE (r= 0.233, p = 0.090); (2)NanoS_FFPE (r= 0.608, p < 0.0001); (3) RNA-Acc_FFPE (r= 0.175, p = 0.21); (4) t-RNA_FFPE (r=−0.237, p = 0.085); (5) and t-RNA (r= −0.012, p = 0.93). These results suggest that only NanoString has successful FF to FFPE translation. The subsequentremoval of identified “problematic” samples (n= 15) and genes (n= 2) further improves the correlations of Affy_FF with three of the five technologies: Affy_FFPE (r= 0.672, p < 0.0001); NanoS_FFPE (r= 0.738, p < 0.0001); and RNA-Acc_FFPE (r= 0.483, p = 0.002). Conclusions: Of the five technology platforms tested, Nano String technology provides a more faithful translation of the RAS pathway gene expression signature from FF to FFPE than the Affymetrix GeneChip and multiple RNASeq technologies. Moreover, Nano String was the most forgiving technology in the analysis of samples with presumably poor RNA quality. Using this approach, the RAS signature score may now be reasonably applied to FFPE clinical samples

Large scale physiological readjustment during growth enables rapid, comprehensive and inexpensive systems analysis

Abstract Background Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable. Results We have discovered in the model organism Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control. Conclusions Simple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems

Real Time Animation of Virtual Humans: A Trade-off Between Naturalness and Control

Virtual humans are employed in many interactive applications using 3D virtual environments, including (serious) games. The motion of such virtual humans should look realistic (or ‘natural’) and allow interaction with the surroundings and other (virtual) humans. Current animation techniques differ in the trade-off they offer between motion naturalness and the control that can be exerted over the motion. We show mechanisms to parametrize, combine (on different body parts) and concatenate motions generated by different animation techniques. We discuss several aspects of motion naturalness and show how it can be evaluated. We conclude by showing the promise of combinations of different animation paradigms to enhance both naturalness and control

Prevalence of transcription promoters within archaeal operons and coding sequences

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C or C photosynthesis. As part of a multinational effort to introduce C traits into rice to boost crop yield, candidate regulators of C leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C -like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C plants

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis.

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51's localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8(+) cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Implicating genes, pleiotropy, and sexual dimorphism at blood lipid loci through multi-ancestry meta-analysis

Publisher Copyright: © 2022, The Author(s).Background: Genetic variants within nearly 1000 loci are known to contribute to modulation of blood lipid levels. However, the biological pathways underlying these associations are frequently unknown, limiting understanding of these findings and hindering downstream translational efforts such as drug target discovery. Results: To expand our understanding of the underlying biological pathways and mechanisms controlling blood lipid levels, we leverage a large multi-ancestry meta-analysis (N = 1,654,960) of blood lipids to prioritize putative causal genes for 2286 lipid associations using six gene prediction approaches. Using phenome-wide association (PheWAS) scans, we identify relationships of genetically predicted lipid levels to other diseases and conditions. We confirm known pleiotropic associations with cardiovascular phenotypes and determine novel associations, notably with cholelithiasis risk. We perform sex-stratified GWAS meta-analysis of lipid levels and show that 3–5% of autosomal lipid-associated loci demonstrate sex-biased effects. Finally, we report 21 novel lipid loci identified on the X chromosome. Many of the sex-biased autosomal and X chromosome lipid loci show pleiotropic associations with sex hormones, emphasizing the role of hormone regulation in lipid metabolism. Conclusions: Taken together, our findings provide insights into the biological mechanisms through which associated variants lead to altered lipid levels and potentially cardiovascular disease risk.Peer reviewe