Large Changes In Expression Research Articles

Cellular Genes Involved in Redox Regulation Are Altered by Inhibitors of Epstein‐Barr Virus Lytic Gene Expression

Epstein‐Barr virus (EBV), a member of the herpesvirus family, causes infectious mononucleosis and is associated with human cancers, such as Burkitt and Hodgkin lymphomas, gastric carcinoma, and nasopharyngeal carcinoma. EBV establishes a latent infection in B cells, but must be reactivated into the viral lytic cycle to produce more virions and spread the infection among cells and hosts. The viral lytic cascade is initiated by the expression of two viral genes BZLF1 and BRLF1. The transcription of these viral immediate‐early genes is highly regulated. Reactivation of the viral lytic cycle is triggered by a variety of chemical and environmental stimuli. Chemical lytic inducing agents must alter cellular signaling pathways and transcription factors that control viral lytic gene expression. To discover cellular genes involved in viral reactivation, EBV‐positive cells were treated with activators or inhibitors of the viral lytic cycle. Changes in cellular gene expression were measured by next‐generation RNA sequencing (RNA‐seq). Transcriptome analysis identified large changes in expression of cellular genes involved in the redox status of the cell. Future work will connect these cellular pathways to the control of viral lytic gene expression.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular characterization of carbendazim resistance of Fusarium species complex that causes sugarcane pokkah boeng disease

BackgroundPokkah boeng is one of the most serious and devastating diseases of sugarcane and causes significant loss in cane yield and sugar content. Although carbendazim is widely used to prevent fungal diseases, the molecular basis of Fusarium species complex (FSC) resistance to carbendazim remains unknown.ResultsThe EC50 (fungicide concentration that inhibits 50% of mycelial growth) values of carbendazim for 35 FSC isolates collected in cane growing regions of China were ranged from 0.5097 to 0.6941 μg mL− 1 of active ingredient (a.i.), in an average of 0.5957 μg a.i. mL− 1. Among carbendazim-induced mutant strains, SJ51M (F. verticillioides) had a CTG rather than CAG codon (Q134L) at position 134 of the FVER_09254 gene, whereas in the mutant strain HC30M (F. proliferatum) codon ACA at position 351 of the FPRO_07779 gene was replaced by ATA (T351I). Gene expression profiling analysis was performed for SJ51M and its corresponding wild type strain SJ51, with and without carbendazim treatment. The gene expression patterns in SJ51 and SJ51M changed greatly as evidenced by the detection of 850 differentially expressed genes (DEGs). Functional categorization indicated that genes associated with oxidation-reduction process, ATP binding, integral component of membrane, transmembrane transport and response to stress showed the largest expression changes between SJ51M and SJ51. The expression levels of many genes involved in fungicide resistance, such as detoxification enzymes, drug efflux transporters and response to stress, were up-regulated in SJ51M compared to SJ51 with and without carbendazim treatment.ConclusionFSC was sensitive to carbendazim and had the potential for rapid development of carbendazim resistance. The transcriptome data provided insight into the molecular pathways involved in FSC carbendazim resistance.

Regulatory and sequence evolution in response to selection for improved associative learning ability in Nasonia vitripennis

BackgroundSelection acts on the phenotype, yet only the genotype is inherited. While both the phenotypic and genotypic response to short-term selection can be measured, the link between these is a major unsolved problem in evolutionary biology, in particular for complex behavioural phenotypes.ResultsHere we characterize the genomic and the transcriptomic basis of associative learning ability in the parasitic wasp Nasonia vitripennis and use gene network analysis to link the two. We artificially selected for improved associative learning ability in four independent pairs of lines and identified signatures of selection across the genome. Allele frequency diverged consistently between the selected and control lines in 118 single nucleotide polymorphisms (SNPs), clustering in 51 distinct genomic regions containing 128 genes. The majority of SNPs were found in regulatory regions, suggesting a potential role for gene expression evolution. We therefore sequenced the transcriptomes of selected and control lines and identified 36 consistently differentially expressed transcripts with large changes in expression. None of the differentially expressed genes also showed sequence divergence as a result of selection. Instead, gene network analysis showed many of the genes with consistent allele frequency differences and all of the differentially expressed genes to cluster in a single co-expression network. At a functional level, both genomic and transcriptomic analyses implicated members of gene networks known to be involved in neural plasticity and cognitive processes.ConclusionsTaken together, our results reveal how specific cognitive abilities can readily respond to selection via a complex interplay between regulatory and sequence evolution.

Genome-wide Characterization of Brassica rapa Genes Encoding Serine/arginine-rich Proteins: Expression and Alternative Splicing Events by Abiotic Stresses

Serine/arginine-rich (SR) gene family members can diversify the transcriptome and proteome of eukaryotes by facilitating the alternative splicing (AS) of precursor messenger RNAs. Herein, we investigated the evolutionary dynamics, AS patterns, and expression levels of the Brassica rapa SR (BrSR) gene family in young seedlings treated with abiotic stresses. A comparative genomic analysis identified 25 BrSR genes at 18 Arabidopsis loci and three BrSR-like genes at two Arabidopsis loci. Thirteen of these loci were singletons, while seven loci carried paralogs. All the duplicated pairs were determined to be under purifying selection pressure. The expansion of the BrSR gene family was found to be the result of segmental duplications only. Additionally, the expression levels of 78.6% (22 of 28) and the AS patterns of 60.7% (17 of 28) of the BrSR genes were altered in response to abiotic stresses. Among the analyzed abiotic stresses, oxidative, cold, and heat treatments induced the largest expression changes, while cold and heat stresses caused most AS events. Our findings provide insights into the evolutionary dynamics of BrSR genes following polyploidization events, and provide an important resource for future studies aimed at characterizing the specific function(s) of BrSR genes in plant growth, development, and defense.

Lunapark, a Novel Target to miR-126—Its Potential Role in Maintenance of ER and Glucose Homeostasis

Strong evidence suggests that an adverse in utero and/or early postnatal environment impacts on long-term risk of developing type 2 diabetes. We showed previously that miR-126 is increased in adipose tissue of mouse offspring born to obese mothers which leads to impaired insulin signaling pathway by silencing IRS-1 at the translational level. However, the full spectrum of targets of miR-126 and consequently the consequences of its overexpression for adipose tissue function are unknown. The aim of the current study was therefore to identify novel targets of miR-126 using the proteomic technique, known as Pulsed Stable Isotope Labeling by Amino Acids (pSILAC). 3T3-L1-cells were transfected with miR-126 and their proteome compared to those transfected with a scrambled sequence. We detected 4567 proteins that were translated in adipocytes and of these 401 demonstrated a >1.3 fold decrease following over-expression of miR-126. Bioinformatic analysis revealed that 43 of these contained a miR-126 seed sequence in their 3’un-translated region. This included known miR-126 targets such as IRS-1 and VCAM-1 as well as novel targets. One of the largest changes in expression was observed for Lunapark and through the use of luciferase assays and western blotting we independently confirmed this was a direct target of miR-126. Lunapark is a key component for stabilization of nascent three-way junctions in the endoplasmic reticulum (ER). Consistent with this role we observed altered levels of mTOR and XBP1 in cells treated with miR-126, reflecting the presence of ER stress. Together, the results suggest that overexpression of miR-126 can lead to both ER stress and Insulin Resistance and therefore represent a novel link between two pathways that contribute to development and progression to T2DM. Disclosure J.A. Faria: None. D. Duque Guimaraes: None. L. Pantaleao: None. T.P. Ong: None. A. Ozanne: None.

Pleiotropy and Flux Control in the Indolic Biochemical Pathways in Arabidopsis thaliana

Metabolic pathways are a series of highly interconnected networks. Predicting how changes in one pathway influence the rest of metabolism is a challenge. Small changes in the expression of one gene in a single pathway may have far reaching pleiotropic effects across the genome or, alternatively, large changes in expression of one gene may be buffered and have only local effects. However, taking into account pathway properties of an enzyme (including the connectedness to other parts of metabolism or the amount of control an enzyme has over pathway production) may lead to predictable impacts on gene expression across the genome (e.g. perturbations in highly connected enzymes to influence a wide range of pathways). To address this question, we are studying the indolic glucosinolate pathway in Arabidopsis thaliana. Glucosinolates are herbivore defense compounds produced by relatives of Arabidopsis and protect the plant against generalist herbivores. The indolic glucosinolate pathway is also connected to the incredibly important plant hormone pathway, auxin, in that the pathways share a product (indole‐3‐acetaldoxime). Auxin is a growth hormone involved in nearly every step of plant development, thus we predict that some changes to the indolic pathway will produce only local effects while some changes may produce wide‐ranging effects. To investigate the influence of pathway position and connectedness on global gene expression and glucosinolate production we created gene expression knockdown lines in every gene in the indolic glucosinolate pathway. By monitoring indole glucosinolate pathway output with High Performance Liquid Chromatography (HPLC) and measuring global gene expression with RNAseq, we can study how changing gene expression within a pathway influences the output of that pathway and gene expression across the genome.Support or Funding InformationSD BRIN P20GM103443SD EPSCOR IIA‐1355423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Coordinated microRNA/mRNA expression profiles reveal a putative mechanism of corneal epithelial cell transdifferentiation from skin epidermal stem cells.

Skin epidermal stem cells (SESCs), which share a single origin with corneal epithelial cells (CECs), are considered to be one of the most ideal seed cells for the construction of tissue engineered corneas. However, the mechanism underlying the transdifferentiation of SESCs to CECs has not been fully elucidated. In the present study, to identify critical microRNAs (miRNAs/miRs) and genes that regulate the transdifferentiation of SESCs to CECs, SESCs and CECs were collected from sheep and used for small RNA sequencing and mRNA microarray analyses. Among the differentially expressed miRNAs and genes, 36 miRNAs were downregulated and 123 genes were upregulated in the CECs compared with those in the SESCs. miR-10b exhibited the largest change in expression between the cell types. Target genes of the 36 downregulated miRNAs were predicted and a computational approach demonstrated that these target genes may be involved in several signaling pathways, including the 'PI3K signaling pathway', the 'Wnt signaling pathway' and the 'MAPK signaling pathway', as well as in 'focal adhesion'. Comparison of these target genes to the 123 upregulated genes identified 43 intersection genes. A regulatory network of these 43 intersection genes and its correlative miRNAs were constructed, and three genes (dedicator of cytokinesis 9, neuronal differentiation 1 and activated leukocyte cell adhesion molecule) were found to have high interaction frequencies. The expression levels of 7 randomly selected miRNAs and the 3 intersection genes were further validated by reverse transcription-quantitative polymerase chain reaction. It was found that miR-10b, the Wnt signaling pathway and the 3 intersection genes may act together and serve a critical role in the transdifferentiation process. This study identified miRNAs and genes that were expressed in SESCs and CECs that may assist in uncovering its underlying molecular mechanism, as well as promote corneal tissue engineering using epidermal stem cells for clinical applications.

Timing and localization of human dystrophin isoform expression provide insights into the cognitive phenotype of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscular dystrophy with high incidence of learning and behavioural problems and is associated with neurodevelopmental disorders. To gain more insights into the role of dystrophin in this cognitive phenotype, we performed a comprehensive analysis of the expression patterns of dystrophin isoforms across human brain development, using unique transcriptomic data from Allen Human Brain and BrainSpan atlases. Dystrophin isoforms show large changes in expression through life with pronounced differences between the foetal and adult human brain. The Dp140 isoform was expressed in the cerebral cortex only in foetal life stages, while in the cerebellum it was also expressed postnatally. The Purkinje isoform Dp427p was virtually absent. The expression of dystrophin isoforms was significantly associated with genes implicated in neurodevelopmental disorders, like autism spectrum disorders or attention-deficit hyper-activity disorders, which are known to be associated to DMD. We also identified relevant functional associations of the different isoforms, like an association with axon guidance or neuron differentiation during early development. Our results point to the crucial role of several dystrophin isoforms in the development and function of the human brain.

Contrasting Frequencies and Effects of cis- and trans-Regulatory Mutations Affecting Gene Expression.

Heritable differences in gene expression are caused by mutations in DNA sequences encoding cis-regulatory elements and trans-regulatory factors. These two classes of regulatory change differ in their relative contributions to expression differences in natural populations because of the combined effects of mutation and natural selection. Here, we investigate how new mutations create the regulatory variation upon which natural selection acts by quantifying the frequencies and effects of hundreds of new cis- and trans-acting mutations altering activity of the TDH3 promoter in the yeast Saccharomyces cerevisiae in the absence of natural selection. We find that cis-regulatory mutations have larger effects on expression than trans-regulatory mutations and that while trans-regulatory mutations are more common overall, cis- and trans-regulatory changes in expression are equally abundant when only the largest changes in expression are considered. In addition, we find that cis-regulatory mutations are skewed toward decreased expression while trans-regulatory mutations are skewed toward increased expression. We also measure the effects of cis- and trans-regulatory mutations on the variability in gene expression among genetically identical cells, a property of gene expression known as expression noise, finding that trans-regulatory mutations are much more likely to decrease expression noise than cis-regulatory mutations. Because new mutations are the raw material upon which natural selection acts, these differences in the frequencies and effects of cis- and trans-regulatory mutations should be considered in models of regulatory evolution.

Multiplexed, Proteome-Wide Protein Expression Profiling: Yeast Deubiquitylating Enzyme Knockout Strains.

Characterizing a protein's function often requires a description of the cellular state in its absence. Multiplexing in mass spectrometry-based proteomics has now achieved the ability to globally measure protein expression levels in yeast from 10 cell states simultaneously. We applied this approach to quantify expression differences in wild type and nine deubiquitylating enzyme (DUB) knockout strains with the goal of creating "information networks" that might provide deeper, mechanistic insights into a protein's biological role. In total, more than 3700 proteins were quantified with high reproducibility across three biological replicates (30 samples in all). DUB mutants demonstrated different proteomics profiles, consistent with distinct roles for each family member. These included differences in total ubiquitin levels and specific chain linkages. Moreover, specific expression changes suggested novel functions for several DUB family members. For instance, the ubp3Δ mutant showed large expression changes for members of the cytochrome C oxidase complex, consistent with a role for Ubp3 in mitochondrial regulation. Several DUBs also showed broad expression changes for phosphate transporters as well as other components of the inorganic phosphate signaling pathway, suggesting a role for these DUBs in regulating phosphate metabolism. These data highlight the potential of multiplexed proteome-wide analyses for biological investigation and provide a framework for further study of the DUB family. Our methods are readily applicable to the entire collection of yeast deletion mutants and may help facilitate systematic analysis of yeast and other organisms.

A RNA-Seq Analysis of the Rat Supraoptic Nucleus Transcriptome: Effects of Salt Loading on Gene Expression.

Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) are highly specialized to release large amounts of arginine vasopressin (Avp) or oxytocin (Oxt) into the blood stream and play critical roles in the regulation of body fluid homeostasis. The MCNs are osmosensory neurons and are excited by exposure to hypertonic solutions and inhibited by hypotonic solutions. The MCNs respond to systemic hypertonic and hypotonic stimulation with large changes in the expression of their Avp and Oxt genes, and microarray studies have shown that these osmotic perturbations also cause large changes in global gene expression in the HNS. In this paper, we examine gene expression in the rat supraoptic nucleus (SON) under normosmotic and chronic salt-loading SL) conditions by the first time using “new-generation”, RNA sequencing (RNA-Seq) methods. We reliably detect 9,709 genes as present in the SON by RNA-Seq, and 552 of these genes were changed in expression as a result of chronic SL. These genes reflect diverse functions, and 42 of these are involved in either transcriptional or translational processes. In addition, we compare the SON transcriptomes resolved by RNA-Seq methods with the SON transcriptomes determined by Affymetrix microarray methods in rats under the same osmotic conditions, and find that there are 6,466 genes present in the SON that are represented in both data sets, although 1,040 of the expressed genes were found only in the microarray data, and 2,762 of the expressed genes are selectively found in the RNA-Seq data and not the microarray data. These data provide the research community a comprehensive view of the transcriptome in the SON under normosmotic conditions and the changes in specific gene expression evoked by salt loading.

Gene expression in the mammary gland of the tammar wallaby during the lactation cycle reveals conserved mechanisms regulating mammalian lactation.

The tammar wallaby (Macropus eugenii), an Australian marsupial, has evolved a different lactation strategy compared with eutherian mammals, making it a valuable comparative model for lactation studies. The tammar mammary gland was investigated for changes in gene expression during key stages of the lactation cycle using microarrays. Differentially regulated genes were identified, annotated and subsequent gene ontologies, pathways and molecular networks analysed. Major milk-protein gene expression changes during lactation were in accord with changes in milk-protein secretion. However, other gene expression changes included changes in genes affecting mRNA stability, hormone and cytokine signalling and genes for transport and metabolism of amino acids and lipids. Some genes with large changes in expression have poorly known roles in lactation. For instance, SIM2 was upregulated at lactation initiation and may inhibit proliferation and involution of mammary epithelial cells, while FUT8 was upregulated in Phase 3 of lactation and may support the large increase in milk volume that occurs at this point in the lactation cycle. This pattern of regulation has not previously been reported and suggests that these genes may play a crucial regulatory role in marsupial milk production and are likely to play a related role in other mammals.

Retinal transcriptome profiling at transcription start sites: a cap analysis of gene expression early after axonal injury.

BackgroundGlaucoma is characterized by progressive loss of the visual field and death of retinal ganglion cells (RGCs), a process that is mediated, in part, by axonal injury. However, the molecular pathomechanisms linking RGC death and axonal injury remain largely unknown. Here, we examined these mechanisms with a cap analysis of gene expression (CAGE), which allows the comprehensive quantification of transcription initiation across the entire genome. We aimed to identify changes in gene expression patterns and to predict the resulting alterations in the protein network in the early phases of axonal injury in mice.ResultsWe performed optic nerve crush (ONC) in mice to model axonal injury. Two days after ONC, the retinas were isolated, RNA was extracted, and a CAGE library was constructed and sequenced. CAGE data for ONC eyes and sham-treated eyes was compared, revealing 180 differentially expressed genes. Among them, the Bcat1 gene, involved in the catabolism of branched-chain amino acid transaminase, showed the largest change in expression (log2 fold-change = 6.70). In some differentially expressed genes, alternative transcription start sites were observed in the ONC eyes, highlighting the dynamism of transcription initiation in a state of disease. In silico pathway analysis predicted that ATF4 was the most significant upstream regulator orchestrating pathological processes after ONC. Its downstream candidate targets included Ddit3, which is known to induce cell death under endoplasmic reticulum stress. In addition, a regulatory network comprising IFNG, P38 MAPK, and TP53 was predicted to be involved in the induction of cell death.ConclusionThrough CAGE, we have identified differentially expressed genes that may account for the link between axonal injury and RGC death. Furthermore, an in silico pathway analysis provided a global view of alterations in the networks of key regulators of biological pathways that presumably take place in ONC. We thus believe that our study serves as a valuable resource to understand the molecular processes that define axonal injury-driven RGC death.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-982) contains supplementary material, which is available to authorized users.

Altering O-Linked β-N-Acetylglucosamine Cycling Disrupts Mitochondrial Function

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.

Transcriptome profiling of mice testes following low dose irradiation

BackgroundRadiotherapy is used routinely to treat testicular cancer. Testicular cells vary in radio-sensitivity and the aim of this study was to investigate cellular and molecular changes caused by low dose irradiation of mice testis and to identify transcripts from different cell types in the adult testis.MethodsTranscriptome profiling was performed on total RNA from testes sampled at various time points (n = 17) after 1 Gy of irradiation. Transcripts displaying large overall expression changes during the time series, but small expression changes between neighbouring time points were selected for further analysis. These transcripts were separated into clusters and their cellular origin was determined. Immunohistochemistry and in silico quantification was further used to study cellular changes post-irradiation (pi).ResultsWe identified a subset of transcripts (n = 988) where changes in expression pi can be explained by changes in cellularity. We separated the transcripts into five unique clusters that we associated with spermatogonia, spermatocytes, early spermatids, late spermatids and somatic cells, respectively. Transcripts in the somatic cell cluster showed large changes in expression pi, mainly caused by changes in cellularity. Further investigations revealed that the low dose irradiation seemed to cause Leydig cell hyperplasia, which contributed to the detected expression changes in the somatic cell cluster.ConclusionsThe five clusters represent gene expression in distinct cell types of the adult testis. We observed large expression changes in the somatic cell profile, which mainly could be attributed to changes in cellularity, but hyperplasia of Leydig cells may also play a role. We speculate that the possible hyperplasia may be caused by lower testosterone production and inadequate inhibin signalling due to missing germ cells.

The Transcriptional Consequences of Somatic Amplifications, Deletions, and Rearrangements in a Human Lung Squamous Cell Carcinoma

Lung cancer causes more deaths, worldwide, than any other cancer. Several histologic subtypes exist. Currently, there is a dearth of targeted therapies for treating one of the main subtypes: squamous cell carcinoma (SCC). As for many cancers, lung SCC karyotypes are often highly anomalous owing to large somatic structural variants, some of which are seen repeatedly in lung SCC, indicating a potential causal association for genes therein. We chose to characterize a lung SCC genome to unprecedented detail and integrate our findings with the concurrently characterized transcriptome. We aimed to ascertain how somatic structural changes affected gene expression within the cell in ways that could confer a pathogenic phenotype. We sequenced the genomes of a lung SCC cell line (LUDLU-1) and its matched lymphocyte cell line (AGLCL) to more than 50x coverage. We also sequenced the transcriptomes of LUDLU-1 and a normal bronchial epithelium cell line (LIMM-NBE1), resulting in more than 600 million aligned reads per sample, including both coding and non-coding RNA (ncRNA), in a strand-directional manner. We also captured small RNA (<30 bp). We discovered significant, but weak, correlations between copy number and expression for protein-coding genes, antisense transcripts, long intergenic ncRNA, and microRNA (miRNA). We found that miRNA undergo the largest change in overall expression pattern between the normal bronchial epithelium and the tumor cell line. We found evidence of transcription across the novel genomic sequence created from six somatic structural variants. For each part of our integrated analysis, we highlight candidate genes that have undergone the largest expression changes.

Response of the goat mammary gland to infection with Staphylococcus aureus revealed by gene expression profiling in milk somatic and white blood cells

BackgroundS. aureus is one of the main pathogens responsible for the intra-mammary infection in dairy ruminants. Although much work has been carried out to understand the complex physiological and cellular events that occur in the mammary gland in response to S. aureus, the protective mechanisms are still poorly understood. The objectives of the present study were to investigate gene expression during the early response of the goat mammary gland to an experimental challenge with S. aureus, in order to better understand the local and systemic response and to compare them in two divergent lines of goat selected for high and low milk somatic cell scores.ResultsNo differences in gene expression were found between high and low SCS (Somatic Cells Score) selection lines. Analysing the two groups together, an expression of 300 genes were found to change from T0 before infection, and T4 at 24 hours and T5 at 30 hours following challenge. In blood derived white blood cells 8 genes showed increased expression between T0 and T5 and 1 gene has reduced expression. The genes showing the greatest increase in expression following challenge (5.65 to 3.16 fold change) play an important role in (i) immune and inflammatory response (NFKB1, TNFAIP6, BASP1, IRF1, PLEK, BATF3); (ii) the regulation of innate resistance to pathogens (PTX3); and (iii) the regulation of cell metabolism (CYTH4, SLC2A6, ARG2). The genes with reduced expression (−1.5 to −2.5 fold) included genes involved in (i) lipid metabolism (ABCG2, FASN), (ii) chemokine, cytokine and intracellular signalling (SPPI), and (iii) cell cytoskeleton and extracellular matrix (KRT19).ConclusionsAnalysis of genes with differential expression following infection showed an inverse relationship between immune response and lipid metabolism in the early response of the mammary gland to the S. aureus challenge. PTX3 showed a large change in expression in both milk and blood, and is therefore a candidate for further studies on immune response associated with mastitis.

Abstract 3686: Single cell assays of heterogeneity and clonality in leukemia

Abstract A range of hypotheses have emerged as to why imatinib (a targeted inhibitor of bcr-abl) seems unable to eliminate the most primitive chronic myeloid leukemia (CML) cells, such as bcr-abl amplification and clonal evolution. The biology of the CML imatinib-resistant cell is of critical importance to understanding why despite high percentages of initial clinical responses to imatinib (&amp;gt;80% of patients in chronic phase at some point obtaining complete cytogenetic remission), resistance, relapse and/or progression occurs in 20-30% of cases and most patients in remission still have residual disease detected by sensitive PCR monitoring of the CML specific bcr-abl transcripts. In acute myeloid leukemia (AML) as well, patients are typically treated with a chemotherapeutic regimen that leads to clinical responses in 80-95% of patients. However, over half of these patients will relapse and eventually die of disease suggesting that a subpopulation of leukemic cells are capable of both surviving treatment and causing regrowth of the tumor. In both diseases, it has not been definitively established what the identity of the cells responsible for resistance and relapse is, nor what combination of mutations, gene expression changes, and alternative splicing events are required for these cells’ resistant phenotype. Assessing changes in gene expression in the bulk samples provides a valuable overall picture of the phenotype of the predominant cell type is or if there are large changes in expression in less frequent cells, but will completely mask changes exhibited by rare cells. One limiting factor in our ability to study more levels of heterogeneity in leukemia subpopulations is the lack of appropriately powerful and sensitive techniques to assay more complex facets of cellular behavior in such small populations. We have developed several multiplexed, high sensitivity analysis techniques and applied them to study multiple facets of cell behavior and identity in rare cell samples, down to the single cell level. By analyzing expression and genotype in smaller samples, direct from cells, we can analyze heterogeneity in expression and alternative splicing, combined with both cell surface markers and the presence of multiple mutations. These innovative techniques have allowed us to study clonality in AML samples in detail via a robust assay that can identify multiple mutations occurring concurrently in single cells. We have also optimized a protocol to simultaneously assay gene expression of 24 in 100-500 cell samples from flow sorted leukemia cell populations. Finally multiplexed assays for alternative splice variant detection in single cells has enabled us to study whether alternative splice variant expression of several genes occurs concurrently in the same cells, or in different cells of the population. With these techniques in hand we have the unique opportunity to begin studying the biology of these diseases from a previously unrealistic angle. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3686. doi:1538-7445.AM2012-3686

Relationship of Cytokine and Regulatory Gene Expression to the Outcomes of Milk and Egg Allergy in an Atopic Cohort (COFAR2)

S A T U R D A Y 94 Relationship of Cytokine and Regulatory Gene Expression to the Outcomes of Milk and Egg Allergy in an Atopic Cohort (COFAR2) S. H. Sicherer, D. Stablein, R. A. Wood, A. W. Burks, A. H. Liu, S. M. Jones, D. M. Fleischer, L. Mayer, R. Lindblad, A. Grishin, H. A. SampsonConsortium of Food Allergy Research. Mt. Sinai School of Medicine, New York, NY, EMMES Corp., Rockville, MD, Johns Hopkins University School of Medicine, Baltimore, MD, Duke, Durham, NC, National Jewish Health, Denver, CO, University of Arkansas for Medical Sciences, Little Rock, AR. RATIONALE: To determine lymphocyte activation biomarkers associated with milk/egg allergy over time. METHODS: Children aged 3-15 months with likely milk/egg allergy and no diagnosed peanut allergywere followed at baseline, 6, 12 and 24months and categorized as egg/milk allergic or tolerant by predefined criteria. Mononuclear cell allergen stimulation screening for CD25, CISH, FOXP3, GATA3, IL-10, IL-4, IFN-gamma and TBET expression using casein and egg white as stimulants were performed at each of the time points. Repeated measures analyses with generalized linear modeling were constructed to determine if gene expression was predictive of the allergy state (allergic or tolerant). RESULTS: The number of children with a confirmed/convincing milk allergy and number tolerant in parentheses among those tested at baseline (n5492), 6 (n5432), 12 (n5420) and 24(n5367) months were: 237(105), 197(137), 197(163) and 145(190), respectively. The corresponding results for egg were: 139(23), 132(35), 129(55) and 99 (117), respectively. The significant (p < 0.05) predictors of allergy based upon current gene expression values were: increased IL4 expression (milk) and decreased GATA3 (egg). Considering change in activation from baseline, risk was associated with a decrease in TBET (milk) and increased IL4 (egg). Tolerance to egg was associated with current decreased CISH, and decreased TBET compared to baseline. However, the predictive capacity of the findingswasminimal, with only a few percentage points of risk being predicted by large changes in expression. CONCLUSIONS: Allergen-stimulated lymphocyte gene expression failed to significantly reflect allergy states over time.

Expression divergence measured by transcriptome sequencing of four yeast species

BackgroundThe evolution of gene expression is a challenging problem in evolutionary biology, for which accurate, well-calibrated measurements and methods are crucial.ResultsWe quantified gene expression with whole-transcriptome sequencing in four diploid, prototrophic strains of Saccharomyces species grown under the same condition to investigate the evolution of gene expression. We found that variation in expression is gene-dependent with large variations in each gene's expression between replicates of the same species. This confounds the identification of genes differentially expressed across species. To address this, we developed a statistical approach to establish significance bounds for inter-species differential expression in RNA-Seq data based on the variance measured across biological replicates. This metric estimates the combined effects of technical and environmental variance, as well as Poisson sampling noise by isolating each component. Despite a paucity of large expression changes, we found a strong correlation between the variance of gene expression change and species divergence (R2 = 0.90).ConclusionWe provide an improved methodology for measuring gene expression changes in evolutionary diverged species using RNA Seq, where experimental artifacts can mimic evolutionary effects.GEO Accession Number: GSE32679