Quantitative Changes In Gene Expression Research Articles

Supplementation of media with gamma-oryzanol as a novel antioxidant to overcome redox imbalance during bovine oocyte maturation invitro.

This study evaluated the effect of supplementing IVM media with γ-oryzanol (ORY), a nutraceutical derived from rice bran oil, on the development of bovine oocytes and hindering the compromising effect of redox imbalance. An invitro model of the bovine cumulus-oocyte complex was used for the evaluation of nuclear maturation and development. Antioxidant activity was investigated by assessing the level of ROS (Reactive Oxygen Species) and GSH (glutathione) in oocytes and quantitative changes in gene expression in matured oocytes and their respective cumulus cells. ORY supplementation increased the proportion of MII oocytes, cleaved embryos, and total blastocysts (p < .05) and was linked to higher and lower levels of intracellular GSH and ROS, respectively (p < .05). The treated oocytes and their respective cumulus-granulosa cells showed a modulation in the expression of genes related to apoptosis (downregulation of BAX and CHOP) and oxidative stress (upregulation of NRF2, CAT, and SOD). Also, relative upregulation of OCT-4 and IGF2R in treated oocytes was concomitant with higher subsequent development in terms of cleavage and total blastocyst rates (p < .05). Based on our findings, it appears that ORY supplementation can improve the nuclear maturation and development of bovine oocytes into blastocysts and augment their enzymatic and non-enzymatic antioxidant systems, maintaining the Redox balance and high enzymatic activity against ROS generation.

Differential Gene Expression Analysis of Non-Small Cell Lung Cancer Samples to Classify Candidate Genes

Differential gene expression is an analysis of gene data, in which the RNA sequence data after next-generation sequencing are to be visualized for any quantitative changes in the levels of the experimental data set. This work aims to derive the transcript statistics on a gene transcript file with a fold change of genes on a normalized scale, in order to identify quantitative changes in gene expression of the difference between the reference genome and Non-Small Cell Lung Cancer (NSCLC) samples. This insight makes a clinical impact in assessing and characterizing candidate genes. The pipeline comprises tuxedo protocol and programming language R with the standard ballgown package. The resultant data set and the plot displays depict the candidate genes in their respective location which are significant in expressing their changes in NSCLC samples. The samples are compared with prominent gene labels of NSCLC samples. The results explain the differential expression of particular samples across samples from both genders.

Single-Cell Transcriptomics of Human Plasma Cells from HLA-Sensitized Patients Reveals Population Heterogeneity and Drug Resistance Adaptations

Kidney transplantation improves the overall survival of end stage renal disease patients but the presence of preformed human leukocyte antigen (HLA)-specific antibodies often precludes finding an acceptable donor. Recent studies demonstrate that bone marrow plasma cells (BMPCs) are a major source of HLA-specific antibodies in highly sensitized transplant candidates. However, therapeutic strategies that target BMPCs and overcome HLA desensitization are only beginning to emerge. Proteasome inhibitor (PI) therapies effectively eliminate BMPCs, reduce HLA-antibody levels and have shown promise as an effective means of enhancing transplantation outcomes. However, PIs are frequently limited by de novo or therapy-induced drug resistance. In addition, BMPCs exhibit variable sensitivity to PI-based therapy. To identify potential mechanisms of drug resistance in BMPCs, we employed single-cell genomic profiling. Single-cell studies provide a powerful tool that allows us to study rare cell populations, such as BMPCs, and intermediate cell states that cannot be resolved at the population level. Here, we have performed single-cell genomic profiling of human BMPCs isolated from highly HLA-sensitized renal transplant candidates prior to and after desensitization therapy with the irreversible PI carfilzomib. HLA-sensitized transplant candidates received CFZ (20, 27, 36 mg/m2) with BM aspirates performed prior to and after CFZ therapy. CD138+BMPCs were isolated to >98% purity and individual cells analyzed using the recently developed method, Iterative Clustering and Guide-gene Selection (ICGS), which utilizes pair-wise correlation of dynamically expressed genes and iterative clustering with pattern-specific guide genes to delineate coherent gene-expression patterns. Strongly and differentially expressed gene sets were clustered and evaluated using gene enrichment, prior knowledge-based networks and ICGS. Each cluster corresponds to one or a group of closely related cell types that represent spatial or temporal distinct states. Single-cell profiling by the drop-seq method analyzed mRNA expression in 2,000 individual BMPCs isolated from the same HLA-sensitized patient prior to and after CFZ therapy. We used CellHarmony, a new bioinformatic approach that allows us to determine the frequency of all CFZ-treated cells in direct reference to the untreated cells and any altered gene expression profiles. In our first comparison of these cells, eight cell clusters were defined (Fig. 1A). Single-cell analysis of BMPCs obtained post-CFZ therapy indicated qualitative and quantitative changes in gene expression as well differences in the proportionality of existing cell clusters. To determine the frequency and expression of these genes in the CFZ-treated samples, we then performed classification of those samples using a k-nearest neighbor approach using all untreated cells as the reference. For example, protein catabolism, nonsense-mediated decay and integrin signaling were increased (clusters 1, 5 and 6) while clusters enriched in IL-12 signaling, interferon signaling and protein translation genes (2, 3 and 7) were reduced. Importantly, the state associated with the mitotic cell cycle was significantly upregulated in BMPCs that survived CFZ. BMPCs were analyzed from additional patients using a comparable approach identified common gene expression sets in pre-treatment samples as well as recurrent differences in post-treatment samples (Fig. 1B).Conclusions: The results presented here demonstrate the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering how cell states respond to therapeutic challenge. Our results indicate a shift toward highly mitotic states in BMPCs that survive CFZ therapy. Importantly, the single cell results also provide internal validation of our prior bulk RNA-seq studies on BMPCs. Our results suggest the feasibility of pathway-based secondary targeting of the recovering cell populations to enhance response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gene expression in the developing embryo and fetus

Determining the stage- and tissue-specific patterns of gene expression shown by the embryo and fetus will provide information about the control of normal development. Identification of alterations in these patterns associated with specific abnormal phenotypes will also be informative regarding the underlying molecular mechanisms. In addition, qualitative and quantitative changes in gene expression that deviate from the norm may provide a potential marker system for predicting future developmental defects, a system that would be particularly useful in preimplantation embryo technologies before recipient transfer. However, there are a number of important issues regarding the interpretation and relevance of many gene expression studies currently undertaken that are often not considered or are ignored. Even when rigorous methodology is applied to detect differences in gene expression, their functional significance is rarely defined. This review discusses the relevance of gene expression changes as diagnostic markers in relation to protein and epigenetic changes and indicates that gene expression studies should be rigorously designed and interpreted to yield meaningful results.

Patterns of alternative splicing in response to cold acclimation in fish.

Phenotypic plasticity is an important aspect of an organism's response to environmental change that often requires the modulation of gene expression. These changes in gene expression can be quantitative, as a result of increases or decreases in the amounts of specific transcripts, or qualitative, as a result of the expression of alternative transcripts from the same gene (e.g. via alternative splicing of pre-mRNAs). Although the role of quantitative changes in gene expression in phenotypic plasticity is well known, relatively few studies have examined the role of qualitative changes. Here, we use skeletal muscle RNA-seq data from Atlantic killifish (Fundulus heteroclitus), threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio) to investigate the extent of qualitative changes in gene expression in response to cold acclimation. Fewer genes demonstrated alternative splicing than differential expression as a result of cold acclimation; however, differences in splicing were detected for 426 to 866 genes depending on species, indicating that large numbers of qualitative changes in gene expression are associated with cold acclimation. Many of these alternatively spliced genes were also differentially expressed, and there was functional enrichment for involvement in muscle contraction among the genes demonstrating qualitative changes in response to cold acclimation. Additionally, there was a common group of 29 genes with cold-acclimation-mediated changes in splicing in all three species, suggesting that there may be a set of genes with expression patterns that respond qualitatively to prolonged exposure to cold temperatures across fishes.

SATrans: New Free Available Software for Annotation of Transcriptome and Functional Analysis of Differentially Expressed Genes.

Recent technological advances have made next-generation sequencing (NGS) a popular and financially accessible technique allowing a broad range of analyses to be done simultaneously. A huge amount of newly generated NGS data, however, require advanced software support to help both in analyzing the data and biologically interpreting the results. In this article, we describe SATrans (Software for Annotation of Transcriptome), a software package providing fast and robust functional annotation of novel sequences obtained from transcriptome sequencing. Moreover, it performs advanced gene ontology analysis of differentially expressed genes, thereby helping to interpret biologically-and in a user-friendly form-the quantitative changes in gene expression. The software is freely available and provides the possibility to work with thousands of sequences using a standard personal computer or notebook running on the Linux operating system.

Resveratrol-cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol-methyl β-cyclodextrin (RV-CD) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV-CD during in vitro oocyte maturation (IVM) or in vitro embryo culture (IVC) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV-CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT-qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV-CD and embryo development and blastocysts gene expression by RT-qPCR were studied. A group without RV-CD (control- ) and a group with cyclodextrin (control+ ) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p<.05). Blastocysts produced by IVC with resveratrol showed that RV-CD could modify the expression of genes related to lipid metabolism (CYP51A1, PNPLA2 and MTORC1) compared with control groups (p<.05). RV-CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts.

Human neuronal changes in brain edema and increased intracranial pressure.

Functional and molecular changes associated with pathophysiological conditions are relatively easily detected based on tissue samples collected from patients. Population specific cellular responses to disease might remain undiscovered in samples taken from organs formed by a multitude of cell types. This is particularly apparent in the human cerebral cortex composed of a yet undefined number of neuron types with a potentially different involvement in disease processes. We combined cellular electrophysiology, anatomy and single cell digital PCR in human neurons identified in situ for the first time to assess mRNA expression and corresponding functional changes in response to edema and increased intracranial pressure. In single pyramidal cells, mRNA copy numbers of AQP1, AQP3, HMOX1, KCNN4, SCN3B and SOD2 increased, while CACNA1B, CRH decreased in edema. In addition, single pyramidal cells increased the copy number of AQP1, HTR5A and KCNS1 mRNAs in response to increased intracranial pressure. In contrast to pyramidal cells, AQP1, HMOX1and KCNN4 remained unchanged in single cell digital PCR performed on fast spiking cells in edema. Corroborating single cell digital PCR results, pharmacological and immunohistochemical results also suggested the presence of KCNN4 encoding the α-subunit of KCa3.1 channels in edema on pyramidal cells, but not on interneurons. We measured the frequency of spontaneous EPSPs on pyramidal cells in both pathophysiological conditions and on fast spiking interneurons in edema and found a significant decrease in each case, which was accompanied by an increase in input resistances on both cell types and by a drop in dendritic spine density on pyramidal cells consistent with a loss of excitatory synapses. Our results identify anatomical and/or physiological changes in human pyramidal and fast spiking cells in edema and increased intracranial pressure revealing cell type specific quantitative changes in gene expression. Some of the edema/increased intracranial pressure modulated and single human pyramidal cell verified gene products identified here might be considered as novel pharmacological targets in cell type specific neuroprotection.

Intermediate Progenitor Cohorts Differentially Generate Cortical Layers and Require Tbr2 for Timely Acquisition of Neuronal Subtype Identity.

Intermediate progenitors (IPs) amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

Transcription in space--environmental vs. genetic effects on differential immune gene expression.

Understanding how organisms adapt to their local environment is one of the key goals in molecular ecology. Adaptation can be achieved through qualitative changes in the coding sequence and/or quantitative changes in gene expression, where the optimal dosage of a gene's product in a given environment is being selected for. Differences in gene expression among populations inhabiting distinct environments can be suggestive of locally adapted gene regulation and have thus been studied in different species (Whitehead & Crawford ; Hodgins-Davis & Townsend ). However, in contrast to a gene's coding sequence, its expression level at a given point in time may depend on various factors, including the current environment. Although critical for understanding the extent of local adaptation, it is usually difficult to disentangle the heritable differences in gene regulation from environmental effects. In this issue of Molecular Ecology, Stutz etal. () describe an experiment in which they reciprocally transplanted three-spined sticklebacks (Gasterosteus aculeatus) between independent pairs of small and large lakes. Their experimental design allows them to attribute differences in gene expression among sticklebacks either to lake of origin or destination lake. Interestingly, they find that translocated sticklebacks show a pattern of gene expression more similar to individuals from the destination lake than to individuals from the lake of origin, suggesting that expression of the targeted genes is more strongly regulated by environmental effects than by genetics. The environmental effect by itself is not entirely surprising; however, the relative extent of it is. Especially when put in the context of local adaptation and population differentiation, as done here, these findings cast a new light onto the heritability of differential gene expression and specifically its relative importance during population divergence and ultimately ecological speciation.

Contextualization Procedure and Modeling of Monocyte Specific TLR Signaling

Innate immunity is the first line of defense against invasion of pathogens. Toll-like receptor (TLR) signaling is involved in a variety of human diseases extending far beyond immune system–related diseases, affecting a number of different tissues and cell-types. Computational models often do not account for cell-type specific differences in signaling networks. Investigation of these differences and its phenotypic implications could increase understanding of cell signaling and processes such as inflammation. The wealth of knowledge for TLR signaling has been recently summarized in a stoichiometric signaling network applicable for constraint-based modeling and analysis (COBRA). COBRA methods have been applied to investigate tissue-specific metabolism using omics data integration. Comparable approaches have not been conducted using signaling networks. In this study, we present ihsTLRv2, an updated TLR signaling network accounting for the association of 314 genes with 558 network reactions. We present a mapping procedure for transcriptomic data onto signaling networks and demonstrate the generation of a monocyte-specific TLR network. The generated monocyte network is characterized through expression of a specific set of isozymes rather than reduction of pathway contents. While further tailoring the network to a specific stimulation condition, we observed that the quantitative changes in gene expression due to LPS stimulation affected the tightly connected set of genes. Differential expression influenced about one third of the entire TLR signaling network, in particular, NF-B activation. Thus, a cell-type and condition-specific signaling network can provide functional insight into signaling cascades. Furthermore, we demonstrate the energy dependence of TLR signaling pathways in monocytes.

Expression and Putative Function of Innate Immunity Genes under in situ Conditions in the Symbiotic Hydrothermal Vent Tubeworm Ridgeia piscesae

The relationships between hydrothermal vent tubeworms and sulfide-oxidizing bacteria have served as model associations for understanding chemoautotrophy and endosymbiosis. Numerous studies have focused on the physiological and biochemical adaptations that enable these symbioses to sustain some of the highest recorded carbon fixation rates ever measured. However, far fewer studies have explored the molecular mechanisms underlying the regulation of host and symbiont interactions, specifically those mediated by the innate immune system of the host. To that end, we conducted a series of studies where we maintained the tubeworm, Ridgeia piscesae, in high-pressure aquaria and examined global and quantitative changes in gene expression via high-throughput transcriptomics and quantitative real-time PCR (qPCR). We analyzed over 32,000 full-length expressed sequence tags as well as 26 Mb of transcript sequences from the trophosome (the organ that houses the endosymbiotic bacteria) and the plume (the gas exchange organ in contact with the free-living microbial community). R. piscesae maintained under conditions that promote chemoautotrophy expressed a number of putative cell signaling and innate immunity genes, including pattern recognition receptors (PRRs), often associated with recognizing microbe-associated molecular patterns (MAMPs). Eighteen genes involved with innate immunity, cell signaling, cell stress and metabolite exchange were further analyzed using qPCR. PRRs, including five peptidoglycan recognition proteins and a Toll-like receptor, were expressed significantly higher in the trophosome compared to the plume. Although PRRs are often associated with mediating host responses to infection by pathogens, the differences in expression between the plume and trophosome also implicate similar mechanisms of microbial recognition in interactions between the host and symbiont. We posit that regulation of this association involves a molecular “dialogue” between the partners that includes interactions between the host’s innate immune system and the symbiont.

A New Level of Complexity

The discovery of the regulatory role of noncoding RNAs, and micro (mi)RNAs in particular, has added a new layer of complexity to our understanding of cardiovascular development. miRNAs regulate and modulate various steps of cardiovascular morphogenesis, cell proliferation, differentiation, and phenotype modulation. miRNAs simultaneously regulate multiple targets, and many miRNAs can bind to the same target, allowing for a complex pattern of regulation of gene expression. miRNA families are continuously added during evolution paralleling the increased complexity of the cardiovascular system in vertebrates compared with invertebrates. Several lines of evidence suggest that the appearance of miRNAs is at least in part responsible for the formation of complex organ systems and stable regulatory mechanisms in vertebrates. We review the current understanding of miRNAs during cardiovascular development. Further progress in this area will help to decipher quantitative changes in gene expression that provide robustness to cellular phenotypes and regulatory options to diseases processes. miRNAs might also provide clues to better understand congenital heart defects, which are the most common birth defects in human newborns.

Multifactorial Origins of Heart and Gut Defects in nipbl-Deficient Zebrafish, a Model of Cornelia de Lange Syndrome

Author SummaryAlthough best known for its role in chromatid cohesion, cohesin is increasingly seen as a regulator of gene expression. In Cornelia de Lange Syndrome (CdLS), partial deficiency for NIPBL, which encodes a cohesin regulator, is associated with small changes in the expression of many genes (similar effects are seen in Nipbl-deficient mice and flies). Are such changes responsible for pervasive developmental defects in CdLS? To address this, we used morpholino oligonucleotides to quantitatively reduce levels of Nipbl protein and Nipbl target genes in zebrafish embryos. Combined knockdown of both zebrafish nipbl genes produced heart and gut defects with similarities to those observed in CdLS. Nipbl-deficient embryos showed quantitative changes in the expression of several genes involved in the specification of endoderm, which both gives rise to gut and provides a substrate for cardiac precursor migration, as well as genes that regulate left-right asymmetry. Functional studies of these putative targets suggest that changes in their expression collectively, and in some cases synergistically, contribute to the observed phenotypes. These findings suggest that birth defects in CdLS result from combinatorial, quantitative effects of NIPBL on gene expression, and suggest that cardiac and visceral organ defects in CdLS arise during early embryonic development.

Genomics of ADME gene expression: mapping expression quantitative trait loci relevant for absorption, distribution, metabolism and excretion of drugs in human liver

Expression quantitative trait loci (eQTL) analysis is a powerful approach toward identifying genetic loci associated with quantitative changes in gene expression. We applied genome-wide association analysis to a data set of >300 000 single-nucleotide polymorphisms and >48 000 mRNA expression phenotypes obtained by Illumina microarray profiling of 149 human surgical liver samples obtained from Caucasian donors with detailed medical documentation. Of 1226 significant associations, only 200 were validated when comparing with a previously published similar study. Potential explanations for low replication rate include differences in microarray platforms, statistical modeling, covariates considered and origin and collection procedures of tissues. Focused analysis revealed a subset of 95 associations related to absorption, distribution, metabolism and excretion of drugs. Of these, 21 were true replications and 74 were newly discovered associations in enzymes, transporters, transcriptional regulators and other genes. This study extends our knowledge about the genetics of inter-individual variability of gene expression with particular emphasis on pharmacogenomics.

Aneuploidy Causes Tissue-Specific Qualitative Changes in Global Gene Expression Patterns in Maize

Segmental aneuploidy refers to the relative excess or deficiency of specific chromosome regions. This condition results in gene dosage imbalance and often causes severe phenotypic alterations in plants and animals. The mechanisms by which gene dosage imbalance affects gene expression and phenotype are not completely clear. The effects of aneuploidy on the transcriptome may depend on the types of cells analyzed and on the developmental stage. We performed global gene expression profiling to determine the effects of segmental aneuploidy on gene expression levels in two different maize (Zea mays) tissues and a detailed analysis of expression of 30 genes affected by aneuploidy in multiple maize tissues. Different maize tissues varied in the frequency at which genes located outside of the aneuploid regions are positively or negatively regulated as well as in the degree of gene dosage compensation. Multiple genes demonstrated qualitative changes in gene expression due to aneuploidy, when the gene became ectopically expressed or completely silenced in aneuploids relative to wild-type plants. Our data strongly suggested that quantitative changes in gene expression at developmental transition points caused by variation in gene copy number progressed through tissue development and resulted in stable qualitative changes in gene expression patterns. Thus, aneuploidy in maize results in alterations of gene expression patterns that differ between tissues and developmental stages of maize seedlings.

Histopathological and molecular evaluation of Holstein-Friesian cows postpartum: Toward an improved understanding of uterine innate immunity

Bovine uterine disease reduces milk yield, impairs fertility and has implications for animal welfare. During involution, the uterus is usually exposed to multiple potential bacterial pathogens which are cleared by successful orchestration of the local inflammatory response. Unsuccessful resolution leads to the development of disease. The aim of this study was to characterize the local innate immune response in the uterus during physiological involution using histopathological and molecular analyses in 9 cows, 2 weeks after calving (early postpartum, EPP), and 4 cows, 9 weeks after calving (late postpartum, LPP). Uterine biopsies taken from each cow were classified by histopathology, and RNA was extracted for molecular analysis. Two EPP cows were classified with a mild, 5 with a moderate and 2 with a severe inflammatory response. Relative gene expression analysis was then performed using quantitative real-time PCR (qRT-PCR) and specific primers for genes encoding Toll-like receptors (TLRs), chemokines, cytokines, acute phase proteins (APPs) and antimicrobial peptides (AMPs). TLR4, transcription factor NFKB1 and the inflammatory cytokines IFNG, IL1A, IL6, IL8, IL12A were all significantly increased in EPP cows ( P < 0.05). Increase in HP, SAA3, TAP and DEFB5 genes was particularly marked in cows with severe inflammation. These results reveal evidence of an inflammatory uterine environment in the early postpartum period with significant induction of both AMP and APP genes. Histopathological grades in EPP cows are underpinned by quantitative changes in gene expression. Understanding the molecular mechanisms contributing to uterine immunity in the early postpartum period may identify candidate genes associated with the resolution of inflammation.

Benzyl alcohol and ethanol can enhance the pathogenic potential of clinical Staphylococcus epidermidis strains

Staphylococcus epidermidis is the most frequent cause of health care-associated infections, particularly in neonates and patients with indwelling catheters. The pathogenesis of infections caused by this organism is associated with its ability to form biofilms. We hypothesized that alcohol used in skin disinfectants, as well as preservative in solutions administered through catheters, can enhance biofilm formation by S epidermidis. We performed polymerase chain reaction (PCR) analysis to investigate the prevalence of ica locus in a collection of 169 commensal and clinical S epidermidis strains. Using a microtiter plate assay, we examined the effect of ethanol and benzyl alcohol on biofilm production. Quantitative real-time reverse transcriptase PCR analysis evaluated quantitative changes in gene expression. We found that ica-positive but biofilm-negative or low-grade biofilm-positive S epidermidis strains displayed induction or increase in biofilm production after incubation in media supplemented with both ethanol and benzyl alcohol. The expression of the icaADBC operon was up-regulated in the presence of alcohol. Our results suggest that biofilm production and, therefore, the pathogen potential of S epidermidis can be induced by alcohol. Considering the routine use of alcohol-based skin disinfectants and benzyl alcohol-containing solutions in hospitals, the alcohol-inducible biofilm phenotype of S epidermidis has potentially profound clinical ramifications.

Alterations of the acetylcholinesterase enzyme in the oriental fruit fly Bactrocera dorsalis are correlated with resistance to the organophosphate insecticide fenitrothion

Alterations of the structure and activity of the enzyme acetylcholinesterase (AChE) leading to resistance to organophosphate insecticides have been examined in the oriental fruit fly, Bactrocera dorsalis (Hendel), an economic pest of great economic importance in the Asia-Pacific region. We used affinity chromatography to purify AChE isoenzymes from heads of insects from lines showing the phenotypes of resistance and sensitivity to insecticide treatments. The AChE enzyme from a strain selected for resistance to the insecticide fenitrothion shows substantially lower catalytic efficiency for various substrates and 124-, 373- and 5810-fold less sensitivity to inhibition by paraoxon, eserine and fenitroxon, respectively, compared to that of the fenitrothion susceptible line. Using peptide mass fingerprinting, we also show how specific changes in the structure of the AChE enzymes in these lines relate to the resistant and sensitive alleles of the AChE ( ace) gene characterized previously in this species (described in Hsu, J.-C., Haymer, D.S., Wu, W.-J., Feng, H.-T., 2006. Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides. Insect Biochem. Mol. Biol. 36, 396–402). Polyclonal antibodies specific to the purified isoenzymes and real-time PCR were also used to show that both the amount of the isoenzyme present and the expression levels of the ace genes were not significantly different between the R and S lines, indicating that quantitative changes in gene expression were not significantly contributing to the resistance phenotype. Overall, our results support a direct causal relationship between the mutations previously identified in the ace gene of this species and qualitative alterations of the structure and function of the AChE enzyme as the basis for the resistance phenotype. Our results also provide a basis for further comparisons of insecticide resistance phenomena seen in closely related species, such as Bactrocera oleae, as well as in a wide range of more distantly related insect species.

Gene Profiling on Mixed Embryonic Stem Cell Populations Reveals a Biphasic Role for β-Catenin in Osteogenic Differentiation

The differentiation of embryonic stem cells (ESCs) into osteoblasts is enhanced to 60% when exposed to vitamin D3 (VD3) but leaves a remainder of one half of the cell population unidentified. To increase differentiation outcome, the known osteoinducers retinoic acid (RA) and bone morphogenetic protein-2 (BMP-2) were evaluated. Initial studies using RA and BMP-2 during early osteogenesis in addition to VD3 increased osteogenic yield in the case of RA, but surprisingly decreased osteogenesis when BMP-2 was administered together with VD3 or RA. This paper describes a comprehensive microarray study examining the gene expression profile of differentiating osteoblasts in these mixed ESC populations. In addition to five other families of signaling molecules (insulin growth factors, prostaglandin, follistatin, TGFbeta2, and Wnt molecules), we identified an endogenous expression pattern for BMPs and RA that differed from our previous exogenous administration of these molecules. By mimicking the change in expression of the RA and BMP-2 families with exogenous supplementation at the correct time, it was then possible to increase the number of ESC-derived osteoblasts to 90%. This effect was mediated through alteration in beta-catenin (CatnB) expression levels and nuclear CatnB activity, both of which are modulated by VD3, RA, and BMP-2. Our results suggest that blockage of CatnB activity by VD3 and RA is opposed by induction of CatnB activity through BMP-2 when administered together. Hence, osteoinduction, in vitro, is an intricate process involving both temporal and quantitative changes in gene expression and CatnB activity.