Biology subjects

Clarke, R.

Publications and source records attributed to Clarke, R..

33 records · Page 2Linked to original sources

bioRxiv2020

Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

SUMMARYGenetic manipulation of mammalian cells is instrumental to modern biomedical research but is currently limited by poor capabilities of sequentially controlling multiple manipulations in cells. Currently, either highly multiplexed manipulations can be delivered to populations of cells all at one time, or gene regulatory sequences can be engineered to conditionally activate a few manipulations within individual cells. Here, we provide proof-of-principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 RNA-guided nuclease and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single guide RNA (sgRNA) - like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of genetic manipulations without the need for engineering cell type specific promoters or gene regulatory sequences. With the expanding diversity of available tools that utilize spCas9 to edit, repress or activate genes, this sgRNA-based system provides multiple levels for interfacing with host cell biology. In addition, ability of the system to progress through multiple modules from episomal plasmid DNA makes it suitable for applications sensitive to the presence of heterologous genomic DNA sequences and broadly applicable to biomedical research and mammalian cell engineering.Competing Interest StatementThe following competing interests are declared for RC, ART, HP, MSM, MR, and BJM as shareholders in Cellgorithmics, Inc., RC, HP, MSM, and BJM as cofounders of Cellgorithmics, Inc., and RC, HP, MSM, and BJM inventors on patent application number PCT/US2018/052211.View Full Text

synthetic biology↗

bioRxiv2019

A dormant sub-population expressing interleukin-1 receptor characterises anti-estrogen resistant ALDH+ breast cancer stem cells

Estrogen receptor-positive (ER+) breast tumours are often treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer Stem Cells (CSCs) with high aldehyde dehydrogenase (ALDH) activity (ALDH+ cells) are reported to be enriched following AE treatment. Here we perform in vitro and in vivo functional CSC assays and gene expression analysis to characterise the ALDH+ population in AE resistant metastatic patient samples and an ER+ cell line. We show that the IL1{beta} signalling pathway is activated in ALDH+ cells and data from single cells reveals that AE treatment selects for IL1R1-expressing ALDH+ cells. Importantly, we demonstrate that increased expression of IL1R1 is observed in the tumours of patients treated with AE therapy and predicts for treatment failure. Single-cell gene expression analysis revealed that at least 2 sub-populations exist within the ALDH+ population, one proliferative and one quiescent. Following AE therapy, the quiescent ALDH+IL1R1+ population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Supporting this, analysis of AE resistant dormant tumours reveals significantly increased expression of ALDH1A1, ALDH1A3 and IL1R1 genes. Thus, we propose that targeting of ALDH+IL1R1+ cells will reverse AE resistance, including in patients with minimal residual disease.

cancer biology↗

bioRxiv2019

Integrated Identification of Disease Specific Pathways Using Multi-omics data

MotivationIdentification of biological pathways plays a central role in understanding both human health and diseases. Although much work has previously been done to explore the biological pathways by using single omics data, little effort has been reported using multi-omics data integration, mainly due to methodological and technological limitations. Compared to single omics data, multi-omics data will help identifying disease specific functional pathways with both higher sensitivity and specificity, thus gaining more comprehensive insights into the molecular architecture of disease processes.\n\nResultsIn this paper, we propose two computational approaches that integrate multi-omics data and identify disease-specific biological pathways with high sensitivity and specificity. Applying our methods to an experimental multi-omics data dataset on muscular dystrophy subtypes, we identified disease-specific pathways of high biological plausibility. The developed methodology will likely have a broad impact on improving the molecular characterization of many common diseases.\n\nContactyuewang@vt.edu\n\nSupplementary informationSupplementary information attached.

bioinformatics↗

bioRxiv2019

PAK4 regulates stemness and progression in endocrine resistant ER-positive metastatic breast cancer

Despite the effectiveness of endocrine therapies to treat estrogen receptor-positive (ER+) breast tumours, two thirds of patients will eventually relapse due to de novo or acquired resistance to these agents. Cancer Stem-like Cells (CSCs), a rare cell population within the tumour, accumulate after anti-estrogen treatments and are likely to contribute to their failure. Here we studied the role of p21-activated kinase 4 (PAK4) as a promising target to overcome endocrine resistance and disease progression in ER+ breast cancers. PAK4 predicts for resistance to tamoxifen and poor prognosis in 2 independent cohorts of ER+ tumours. We observed that PAK4 strongly correlates with CSC activity in metastatic patient-derived samples irrespective of breast cancer subtype. However, PAK4-driven mammosphere-forming CSC activity increases alongside progression only in ER+ metastatic samples. PAK4 activity increases in ER+ models during acquired resistance to endocrine therapies. Targeting PAK4 with either CRT PAKi, a small molecule inhibitor of PAK4, or with specific siRNAs abrogates CSC activity/self-renewal in clinical samples and endocrine-resistant cells. Together, our findings establish that PAK4 regulates stemness during disease progression and that its inhibition reverses endocrine resistance in ER+ breast cancers. HighlightsO_LIPAK4 predicts for failure of endocrine therapies and poor prognosis C_LIO_LIPAK4 drives stemness and progression in ER+ metastatic breast cancer C_LIO_LITargeting PAK4 abrogates breast CSC activity and restores sensitivity to endocrine treatments C_LIO_LITargeting PAK4 will improve outcome of ER+ breast cancer patients C_LI List of Abbreviations that appeared in abstractCancer Stem-like Cells (CSCs) p21-activated kinase 4 (PAK4) Estrogen Receptor (ER)

cancer biology↗

bioRxiv2019

The role of SLC7A5 (LAT1) in endocrine therapy resistant breast cancer

Resistance to endocrine therapies remains a major challenge for the successful management of patients with estrogen receptor-positive (ER+) breast cancers. Central to the development of resistance is the adaptive reprogramming of cellular metabolism in response to treatment. Solute carriers (SLCs) play a key role in metabolic reprogramming by transporting sugars, amino acids, and other nutrients and regulating their abundance within the cell and its subcellular organelles. We found 109 SLC mRNAs to be differentially expressed between endocrine sensitive and resistant breast cancer cells. In univariate analyses, 55 of these SLCs were associated with poor outcome in ER+ breast cancer patients. Data from TMT and SILAC studies then led us to focus on SLC7A5 (LAT1). In complex with SLC3A2 (CD98), LAT1 is the primary transporter of large, neutral amino acids including leucine and tyrosine. LAT1 expression is estrogen-regulated in endocrine sensitive cells but this regulation is lost in resistant cells. Pharmacologic inhibition or genetic depletion of LAT1 each suppressed growth in two models of endocrine resistant breast cancer. Autophagy was activated with LAT1 inhibition, but cells failed to degrade p62 showing that flux was blocked. Overexpression of the LAT1 cDNA increased protein synthesis and high LAT1 expression correlated with poor disease-free survival in ER+ breast cancer patients. This study uncovers a novel LAT1 mediated adaptive response that contributes to the development of endocrine resistance. Blocking LAT1 function may offer a new avenue for effective therapeutic intervention against endocrine resistant ER+ breast cancers.

cancer biology↗

bioRxiv2019

Data-driven robust detection of tissue/cell-specific markers

Tissue or cell subtype-specific and differentially-expressed genes (SDEGs) are defined as being differentially expressed in a particular tissue or cell subtype among multiple subtypes. Detecting SDEGs plays a critical rolse in molecularly characterizing and identifying tissue or cell subtypes, and facilitating supervised deconvolution of complex tissues. Unfortunately, classic differential analysis assumes a convenient null hypothesis and associated test statistic that is subtype-non-specific and thus, resulting in a high false positive rate and/or lower detection power with respect to particular subtypes. Here we introduce One-Versus-Everyone Fold Change (OVE-FC) test for detecting SDEGs. To assess the statistical significance of such test, we also propose the scaled test statistic OVE-sFC together with a mixture null distribution model and a tailored permutation scheme. Validated with realistic synthetic data sets on both type 1 error and detection power, OVE-FC/sFC test applied to two benchmark gene expression data sets detects many known and de novo SDEGs. Subsequent supervised deconvolution results, obtained using the SDEGs detected by OVE-FC/sFC test, showed superior performance in deconvolution accuracy when compared with popular peer methods.

bioinformatics↗

bioRxiv2018

Functional Anatomy, Biomechanical Performance Capabilities and Potential Niche of StW 573: an Australopithecus Skeleton (circa 3.67 Ma) From Sterkfontein Member 2, and its significance for The Last Common Ancestor of the African Apes and for Hominin Origins

Abstract (300 words)StW 573, from Sterkfontein Member 2, dated ca 3.67 Ma, is by far the most complete skeleton of an australopith to date. Joint morphology is in many cases closely matched in available elements of Australopithecus anamensis (eg. proximal and distal tibial and humeral joint-surfaces) and there are also close similarities to features of the scapula, in particular, of KSD-VP-1/1 A. afarensis from Woranso-Mille. The closest similarities are, however, to the partial skeleton of StW 431 from Sterkfontein Member 4. When considered together, both StW 573 and StW 431 express an hip joint morphology quite distinct from that of A. africanus Sts14, and a proximal femur of a presumed A. africanus from Jacovec Cavern at Sterkfontein, StW 598. This, and other evidence presented herein, suggests there are two pelvic girdle morphs at Sterkfontein, supporting Clarke (2013) in his recognition of a second species, A. prometheus, containing StW 573 and StW 431. StW 573 is the first hominid skeleton where limb proportions are known unequivocally. It demonstrates that some early hominins, at the time of formation of the Laetoli footprints (3.6 Ma), were large-bodied. with hindlimbs longer than forelimbs. Modelling studies on extant primates indicate that the intermembral index (IMI) of StW 573, low for a non-human great ape, would have substantially enhanced economy of bipedal walking over medium-to-long distances, but that it was still too high for effective walking while load-carrying. It would, however, have somewhat reduced the economy of horizontal climbing, but made Gorilla-like embracing of large tree-trunks less possible. Consideration of both ethnographic evidence from modern indigenous arboreal foragers and modern degeneracy theory cautions against prescriptive interpretations of hand- and foot-function, by confirming that both human-like upright bipedalism and functional capabilities of the hand and foot can be effective in short-distance arboreal locomotion.

paleontology↗

bioRxiv2018

Unsupervised deconvolution of molecular heterogeneity uncovers novel signatures and glia-neuron ratio

Introduction Introduction Results Discussion Methods AUTHOR CONTRIBUTIONS Competing interests Appendix References While the two major types of cells in the brain are known to be glia and neuron, the true ratio of glia to neurons in the brain remains a mystery. One of recent studies using efficient cell counting method provides compelling evidence for 1:1 ratio on four whole human brains 1. The same study also reveals that the ratio of glia to neurons in the brain varies from one region to another, sometimes dramatically, e.g., 3.76:1 in the cerebral cortex versus 1:4.3 in the cerebellum 1,2. However, other scientists have argued that more rigorous studies are needed in which just about every known ...

bioinformatics↗

bioRxiv2018

Histone deacetylase activity is required for Botrylloides leachii whole body regeneration.

The colonial tunicate Botrylloides leachii is exceptional at regenerating from a piece of vascular tunic after loss of all adults from the colony. Previous transcriptome analyses indicate a brief period of healing before regeneration of a new adult (zooid) in as little as 8-10 days. However, there is little understanding of how the resulting changes to gene expression, required to drive regeneration, are initiated and how the overall process is regulated. Rapid changes to gene expression often occur in response to chromatin changes, mediated by histone modifications such as histone acetylation. Here, we investigated a group of key epigenetic modifiers, histone deacetylases (HDAC) that are known to play an important role in many biological processes such as development, healing and regeneration.\n\nThrough our transcriptome data, we identified and quantified the expression levels of HDAC and histone acetyltransferase (HAT) enzymes during whole body regeneration (WBR). To determine if HDAC activity is required for WBR, we inhibited its action using valproic acid (VPA) and Trichostatin A (TSA). HDAC inhibition prevented the final morphological changes normally associated with WBR and resulted in aberrant gene expression. B. leachii genes including Slit2, TGF-{square}, Piwi and Fzd4 all showed altered gene expression upon HDAC inhibition in comparison to the control samples. Additionally, atypical expression of Bl_Piwi was found in immunocytes upon HDAC inhibition.\n\nTogether, these results show that HDAC function, specifically HDAC I/IIa class enzymes, are vital for B. leachii to undergo WBR successfully.

molecular biology↗

bioRxiv2018

Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double strand break (DSB), Cas9 remains stably bound to it. Here we show persistent Cas9 binding blocks access to DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches one direction towards the Cas9-DSB complex. By exploiting these RNA polymerase-Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of PAM sequences and a simple method of improving selection of highly active sgRNA for genome editing.

molecular biology↗

bioRxiv2018

Mathematical modelling of molecular heterogeneity identifies novel markers and subpopulations in complex tumors

Intratumor heterogeneity, as both a major confounding factor and an underexploited information source, is widely implicated as a key driver of drug resistance. While a handful of reports have demonstrated the potential of supervised methods to deconvolute intratumor heterogeneity, these approaches require a priori information on the marker genes or composition of known subpopulations. To address the critical problem of the absence of validated marker genes for many (including novel) subpopulations, we developed convex analysis of mixtures (CAM), a fully unsupervised deconvolution method, for identifying marker genes and subpopulations directly from original mixed molecular expressions.

bioinformatics↗

bioRxiv2018

Metabolomic consequences of genetic inhibition of PCSK9 compared with statin treatment

BackgroundBoth statins and PCSK9 inhibitors lower blood low-density lipoprotein cholesterol (LDL-C) levels to reduce risk of cardiovascular events. To assess potential differences between metabolic effects of these two lipid-lowering therapies, we performed detailed lipid and metabolite profiling of a large randomized statin trial, and compared the results with the effects of genetic inhibition of PCSK9, acting as a naturally occurring trial.\n\nMethods228 circulating metabolic measures were quantified by nuclear magnetic resonance spectroscopy, including lipoprotein subclass concentrations and their lipid composition, fatty acids, and amino acids, for 5,359 individuals (2,659 on treatment) in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) trial at 6-months post-randomization. The corresponding metabolic measures were analyzed in eight population cohorts (N=72,185) using PCSK9 rs11591147 as an unconfounded proxy to mimic the therapeutic effects of PCSK9 inhibitors.\n\nResultsScaled to an equivalent lowering of LDL-C, the effects of genetic inhibition of PCSK9 on 228 metabolic markers were generally consistent with those of statin therapy (R2=0.88). Alterations in lipoprotein lipid composition and fatty acid balance were similar. However, discrepancies were observed for very-low-density lipoprotein (VLDL) lipid measures. For instance, genetic inhibition of PCSK9 showed weaker effects on lowering of VLDL-cholesterol compared with statin therapy (54% vs. 77% reduction, relative to the lowering effect on LDL-C; P=2 x 10-7 for heterogeneity). Genetic inhibition of PCSK9 showed no robust effects on amino acids, ketones, and a marker of inflammation (GlycA); in contrast, statin treatment lowered GlycA levels.\n\nConclusionsGenetic inhibition of PCSK9 results in similar metabolic effects as statin therapy across a detailed lipid and metabolite profile. However, for the same lowering of LDL-C, PCSK9 inhibitors are predicted to be less efficacious than statins at lowering VLDL lipids, which could potentially translate into subtle differences in cardiovascular risk reduction.

epidemiology↗

bioRxiv2018

ChIP-BIT2: a software tool to detect weak binding events using a Bayesian integration approach

Transcription factor binding events play important functional roles in gene regulation. It is, however, a challenging task to detect weak binding events since the ambiguity in differentiation of weak binding signals from background signals. We present a software package, ChIP-BIT2, to identify weak binding events using a Bayesian integration approach. By integrating signals from sample and input ChIP-seq data, ChIP-BIT2 can detect both strong and weak binding events at gene promoter, enhancer or the whole genome effectively. The ChIP-BIT2 package has been extensively tested on ChIP-seq data, demonstrating its wide applicability in ChIP-seq data analysis.\n\nAvailability and ImplementationThe ChIP-BIT2 package is available at http://sourceforge.net/projects/chipbitc/.

bioinformatics↗

bioRxiv2017

Effect of vitamin D supplementation on biomarkers of inflammation and immune function: functional genomics analysis of the BEST-D trial

Vitamin D deficiency has been associated with multiple diseases, but the causal relevance and underlying processes are not fully understood. Elucidating the mechanisms of action of drug treatments in humans is challenging, but application of functional genomic approaches in randomised trials may afford an opportunity to systematically assess molecular responses to treatments. In the Biochemical Efficacy and Safety Trial of Vitamin D (BEST-D), 305 community-dwelling individuals aged over 65 years were randomly allocated to treatment with vitamin D34000 IU, 2000 IU or placebo daily for 12 months. Genome-wide genotypes at baseline, and transcriptome and plasma levels of cytokines (IFN-{gamma}, IL-10, IL-8, IL-6 and TNF-) at baseline and after 12 months, were measured. The trial had >90% power to detect a 2-fold change in gene expression. Allocation to vitamin D for 12-months was associated with 2-fold higher plasma levels of 25-hydroxy-vitamin D (25[OH]D), but had no significant effect on whole-blood gene expression (FDR <5%) or on plasma levels of cytokines compared with placebo. In pre-specified analysis, rs7041 (intron variant, GC) had a significant effect on circulating levels of 25(OH)D in the low dose but not on the placebo or high dose vitamin D regimen. A gene expression quantitative trait locus analysis (eQTL) demonstrated evidence of 31,568 cis-eQTLs (unique SNP-probe pairs) among individuals at baseline and 34,254 after supplementation for 12 months (any dose), but had no significant effect on cis-eQTLs specific to vitamin D supplementation. The trial demonstrates the feasibility of application of functional genomics approaches in randomised trials to assess the effects of vitamin D on immune function.\n\nOne sentence summarySupplementation with high-dose vitamin D in older people for 12 months in a randomised, placebo-controlled trial had no significant effect on gene expression or on plasma concentrations of cytokines.\n\nTrial registrationSRCTN registry (Number 07034656) and the European Clinical Trials Database (EudraCT Number 2011-005763-24).\n\nFundingMedical Research Council, British Heart Foundation, Wellcome Trust, European Research Council and Clinical Trial Service Unit, Nuffield Department of Population Health, University of Oxford, Oxford, United Kingdom\n\nCopyrightOpen access article under the terms of CC BY.

clinical trials↗

bioRxiv2017

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term early risers. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

biochemistry↗