Strategies and tools for preventing neurotoxicity: to test, to predict and how to do it

A change in paradigm is needed in the prevention of toxic effects on the nervous system, moving from its present reliance solely on data from animal testing to a prediction model mostly based on in vitro toxicity testing and in silico modeling. According to the report published by the National Research Council (NRC) of the US National Academies of Science, high-throughput in vitro tests will provide evidence for alterations in"toxicity pathways" as the best possible method of large scale toxicity prediction. The challenges to implement this proposal are enormous, and provide much room for debate. While many efforts address the technical aspects of implementing the vision, many questions around it need also to be addressed. Is the overall strategy the only one to be pursued? How can we move from current to future paradigms? Will we ever be able to reliably model for chronic and developmental neurotoxicity in vitro? This paper summarizes four presentations from a symposium held at the International Neurotoxicology Conference held in Xi"an, China, in June 2011. A. Li reviewed the current guidelines for neurotoxicity and developmental neurotoxicity testing, and discussed the major challenges existing to realize the NCR vision for toxicity testing. J. Llorens reviewed the biology of mammalian toxic avoidance in view of present knowledge on the physiology and molecular biology of the chemical senses, taste and smell. This background information supports the hypothesis that relating in vivo toxicity to chemical epitope descriptors that mimic the chemical encoding performed by the olfactory system may provide a way to the long term future of complete in silico toxicity prediction. S. Ceccatelli reviewed the implementation of rodent and human neural stem cells (NSCs) as models for in vitro toxicity testing that measures parameters such as cell proliferation, differentiation and migration. These appear to be sensitive endpoints that can identify substances with developmental neurotoxic potential. C. Sun ol reviewed the use of primary neuronal cultures in testing for neurotoxicity of environmental pollutants, including the study of the effects of persistent exposures and/or in differentiating cells, which allow recording of effects that can be extrapolated to human developmental neurotoxicity.

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Genome-wide transcriptional responses to shade: Linking shade avoidance and auxin

Plants have the ability to use the composition of incident light as a cue to adapt development and growth to their environment. Arabidopsis thaliana as well as many crops are best adapted to sunny habitats. When subjected to shade, these plants exhibit a variety of physiological responses collectively called shade avoidance syndrome (SAS). It includes increased growth of hypocotyl and petioles, decreased growth rate of cotyledons and reduced branching and crop yield. These responses are mainly mediated by phytochrome photoreceptors, which exist either in an active, far-red light (FR) absorbing or an inactive, red light (R) absorbing isoform. In direct sunlight, the R to FR light (R/FR) ratio is high and converts the phytochromes into their physiologically active state. The phytochromes interact with downstream transcription factors such as PHYTOCHROME INTERACTING FACTOR (PIF), which are subsequently degraded. Light filtered through a canopy is strongly depleted in R, which result in a low R/FR ratio and renders the phytochromes inactive. Protein levels of downstream transcription factors are stabilized, which initiates the expression of shade-induced genes such as HFR1, PIL1 or ATHB-2. In my thesis, I investigated transcriptional responses mediated by the SAS in whole Arabidopsis seedlings. Using microarray and chromatin immunoprecipitation data, we identified genome-wide PIF4 and PIF5 dependent shade regulated gene as well as putative direct target genes of PIF5. This revealed evidence for a direct regulatory link between phytochrome signaling and the growth promoting phytohormone auxin (IAA) at the level of biosynthesis, transport and signaling. Subsequently, it was shown, that free-IAA levels are upregulated in response to shade. It is assumed that shade-induced auxin production takes predominantly place in cotyledons of seedlings. This implies, that IAA is subsequently transported basipetally to the hypocotyl and enhances elongation growth. The importance of auxin transport for growth responses has been established by chemical and genetic approaches. To gain a better understanding of spatio-temporal transcriptional regulation of shade-induce auxin, I generated in a second project, an organ specific high throughput data focusing on cotyledon and hypocotyl of young Arabidopsis seedlings. Interestingly, both organs show an opposite growth regulation by shade. I first investigated the spatio-transcriptional regulation of auxin re- sponsive gene, in order to determine how broad gene expression pattern can be explained by the hypothesized movement of auxin from cotyledons to hypocotyls in shade. The analysis suggests, that several genes are indeed regulated according to our prediction and others are regulated in a more complex manner. In addition, analysis of gene families of auxin biosynthetic and transport components, lead to the identification of essential family members for shade-induced growth re- sponses, which were subsequently experimentally confirmed. Finally, the analysis of expression pattern identified several candidate genes, which possibly explain aspects of the opposite growth response of the different organs.

Molecular insight into heart development and congenital heart disease: An update review from the Arab countries.

Congenital heart defect (CHD) has a major influence on affected individuals as well as on the supportive and associated environment such as the immediate family. Unfortunately, CHD is common worldwide with an incidence of approximately 1% and consequently is a major health concern. The Arab population has a high rate of consanguinity, fertility, birth, and annual population growth, in addition to a high incidence of diabetes mellitus and obesity. All these factors may lead to a higher incidence and prevalence of CHD within the Arab population than in the rest of the world, making CHD of even greater concern. Sadly, most Arab countries lack appropriate public health measures directed toward the control and prevention of congenital malformations and so the importance of CHD within the population remains unknown but is thought to be high. In approximately 85% of CHD patients, the multifactorial theory is considered as the pathologic basis. The genetic risk factors for CHD can be attributed to large chromosomal aberrations, copy number variations (CNV) of particular regions in the chromosome, and gene mutations in specific nuclear transcription pathways and in the genes that are involved in cardiac structure and development. The application of modern molecular biology techniques such as high-throughput nucleotide sequencing and chromosomal array and methylation array all have the potential to reveal more genetic defects linked to CHD. Exploring the genetic defects in CHD pathology will improve our knowledge and understanding about the diverse pathways involved and also about the progression of this disease. Ultimately, this will link to more efficient genetic diagnosis and development of novel preventive therapeutic strategies, as well as gene-targeted clinical management. This review summarizes our current understanding of the molecular basis of normal heart development and the pathophysiology of a wide range of CHD. The risk factors that might account for the high prevalence of CHD within the Arab population and the measures required to be undertaken for conducting research into CHD in Arab countries will also be discussed.

Evaluation of GPRS Radio Link Control Layer in Weak RF Field

General Packet Radio Service (GPRS) mahdollistaa pakettimuotoisen tiedonsiirron GSM-verkossa. Se tarjoaa yhteyden pakettidataverkkoihin, nostaen samalla tiedonsiirtonopeutta radiorajapinnassa. Radioresurssit ovat varattuna vain silloin kun on jotain lähetettävää, tehden täten radioresurssien käytön paljon tehokkaammaksi. Tämä diplomityö keskittyy GPRS protokollaan ja erityisesti sen datapinossa olevaan Radio Link Control (RLC) kerrokseen. RLC-kerros huolehtii GPRS- puhelimen ja tukiaseman välisen yhteyden luotettavuudesta. Työn tavoitteena on tutkia RLC-kerroksen toiminnallisuutta ja sen luotettavuutta heikossa kentässä, sekä selvittää heikon kentän vaikutusta uudelleenlähetyksiin. Työn tuloksena saadaan arvio signaalin voimakkuuden sekä uudelleen lähetysten vaikutuksesta GPRS:n datansiirtonopeuteen. Tämä työ käsittelee myös lyhyesti GSM-järjestelmää, koska lukijan on näin helpompaa ymmärtää myös GPRS-järjestelmän vaatimia teknisiä muutoksia. Tämä diplomityö on tehty osana Nokia Matkapuhelimet Oyj:ssä käynnissä olevaa GPRS tuotekehitysprojektia. Työn tuloksia käytetään testauksen tukena ja niitä on käytetty apuna RLC-kerroksen luotettavuustestauksen suunnittelussa.

Production Control Principles in Printed Wiring Board Fabrication

Työn tavoitteena oli kuvata piirilevyvalmistaja Aspocomp Oy:n Espoon tehtaan tämän hetkinen tuotannonohjausperiaate ja tunnistaa siinä esiintyvät puutteet sekä kehittää vaihtoehtoinen tuotannonohjausperiaate piirilevyvalmistukseen. Vaihtoehtoisen ohjausperiaatteen lähtökohtana oli tuotannonohjauksen sopeuttaminen vaativaan ja jatkuvasti muuttuvaan liiketoimintaympäristöön. Työn teoreettinen osa keskittyi tuotannonohjauksen eri lähestymistapoihin. Kirjallisuuskatsauksessa esitetään eri tuotannonohjausperiaatteiden keskeiset sisällöt, jotka muodostavat rungon toimivalle tuotannonohjauskäytännölle. Työn kokeellinen osa keskittyi Espoon piirilevytehtaan tuotannonohjausperiaatteen selvittämiseen. Espoon piirilevytehtaan nykyisessä tuotannonohjausperiaatteessa havaittujen ongelmakohtien ja liiketoimintaympäristön vaatimusten perusteella kehitettiin vaihtoehtoinen tuotannonohjaustapa. Vaihtoehtoisen tuotannonohjaustavan päämääränä oli läpimenoajan lyhentäminen sekä tuotannon parempi hallittavuus. Vaihtoehtoinen toimintamalli tavoitteiden saavuttamiseksi perustuu pullonkaulateoriaan, jossa keskeisin muutos nykyiseen toimintamalliin oli puolivalmisteiden varastointi toimitusajan lyhentämiseksi sekä tuotantovolyymin heilahdusten vaikutusten vähentämiseksi. Työn kokeellisessa osassa ilmeni, että kysynnän muutokset ja kapasiteetin suunnittelun puute aiheuttivat ongelmia piirilevytehtaan tuotannonohjauksessa.

Quantitative proteomics of heat-treated human cells show an across-the-board mild depletion of housekeeping proteins to massively accumulate few HSPs.

Classic semiquantitative proteomic methods have shown that all organisms respond to a mild heat shock by an apparent massive accumulation of a small set of proteins, named heat-shock proteins (HSPs) and a concomitant slowing down in the synthesis of the other proteins. Yet unexplained, the increased levels of HSP messenger RNAs (mRNAs) may exceed 100 times the ensuing relative levels of HSP proteins. We used here high-throughput quantitative proteomics and targeted mRNA quantification to estimate in human cell cultures the mass and copy numbers of the most abundant proteins that become significantly accumulated, depleted, or unchanged during and following 4 h at 41 °C, which we define as mild heat shock. This treatment caused a minor across-the-board mass loss in many housekeeping proteins, which was matched by a mass gain in a few HSPs, predominantly cytosolic HSPCs (HSP90s) and HSPA8 (HSC70). As the mRNAs of the heat-depleted proteins were not significantly degraded and less ribosomes were recruited by excess new HSP mRNAs, the mild depletion of the many housekeeping proteins during heat shock was attributed to their slower replenishment. This differential protein expression pattern was reproduced by isothermal treatments with Hsp90 inhibitors. Unexpectedly, heat-treated cells accumulated 55 times more new molecules of HSPA8 (HSC70) than of the acknowledged heat-inducible isoform HSPA1A (HSP70), implying that when expressed as net copy number differences, rather than as mere "fold change" ratios, new biologically relevant information can be extracted from quantitative proteomic data. Raw data are available via ProteomeXchange with identifier PXD001666.

COMPASS identifies T-cell subsets correlated with clinical outcomes.

Advances in flow cytometry and other single-cell technologies have enabled high-dimensional, high-throughput measurements of individual cells as well as the interrogation of cell population heterogeneity. However, in many instances, computational tools to analyze the wealth of data generated by these technologies are lacking. Here, we present a computational framework for unbiased combinatorial polyfunctionality analysis of antigen-specific T-cell subsets (COMPASS). COMPASS uses a Bayesian hierarchical framework to model all observed cell subsets and select those most likely to have antigen-specific responses. Cell-subset responses are quantified by posterior probabilities, and human subject-level responses are quantified by two summary statistics that describe the quality of an individual's polyfunctional response and can be correlated directly with clinical outcome. Using three clinical data sets of cytokine production, we demonstrate how COMPASS improves characterization of antigen-specific T cells and reveals cellular 'correlates of protection/immunity' in the RV144 HIV vaccine efficacy trial that are missed by other methods. COMPASS is available as open-source software.

Tuotannon läpäisyajan analyysi

Diplomityö on tapaustutkimus, jossa analysoidaan yrityksen tilausohjautuvan massatuotannon läpimenoon vaikuttavia tekijöitä huippukuormituksen aikana. Tavoitteena oli selvittää päätuotantolinjan yksittäisten osien kapasiteetin käyttösuhdetta pienentävät ja koko tuotantoketjun läpäisyaikaan vaikuttavat tekijät, kapasiteetin käyttösuhdetta pienentävien tekijöiden poistamiseksi tarvittavat toimenpiteet ja teoreettinen maksimikapasiteetti näiden toimenpiteiden suorittamisen jälkeen. Selvitystä käytettiin tukimateriaalina päätettäessä tulevaisuuden laiteinvestoinneista. Projektin tiedonkeruun menetelminä toteutettiin osallistuvaa havainnointia, tehtiin aikamittauksia sekä kokeiltiin erilaisia tuotannonohjauksen järjestelyjä. Kerättyjen tietojen pohjalta laadittiin arviolaskelmia kapasiteetin käyttösuhteen määrittämiseksi sekä arvioitiin eri muuttujien vaikutusta. Tutkimuksen perusteella löydettiin kustakin päätuotantolinjan työvaiheesta kapasiteetin käyttösuhdetta vähentäviä tekijöitä ja laadittiin parannusehdotuksia. Esimerkkeinä työaikajärjestelyt ja käyttömiesten toimenkuvaan kuuluvien tehtävien järjestelyt, sekä kunnossapidon, tuotannonohjauksen ja tiedonkulun kehittäminen. Tutkimuksessa esitettyjen parannusehdotusten, kysynnän kehitysennusteiden ja suunnitellun laitteistoinvestoinnin toteutumisen myötä tuotannon pullonkaulatyövaihe siirtyy tuotantoketjussa seuraavaan vaiheeseen. Tämän huomioiminen investointipäätöksissä on oleellista tasaisesti virtaavan tuotannon aikaansaamiseksi.

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining tilling mutant populations.

Background: TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. Results: Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. Conclusions: This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Quantitative proteomics in the characterization of T helper lymphocyte differentiation

The term proteome is used to define the complete set of proteins expressed in cells or tissues of an organism at a certain timepoint. Respectively, proteomics is used to describe the methods, which are used to study such proteomes. These methods include chromatographic and electrophoretic techniques for protein or peptide fractionation, mass spectrometry for their identification, and use of computational methods to assist the complicated data analysis. A primary aim in this Ph.D. thesis was to set-up, optimize, and develop proteomics methods for analysing proteins extracted from T-helper (Th) lymphocytes. First, high-throughput LC-MS/MS and ICAT labeling methods were set-up and optimized for analysing the microsomal fraction proteins extracted from Th lymphocytes. Later, iTRAQ method was optimized to study cytokine regulated protein expression in the nuclei of Th lymphocytes. High-throughput LC-MS/MS analyses, like ICAT and iTRAQ, produce large quantities of data and robust software and data analysis pipelines are needed. Therefore, different software programs used for analysing such data were evaluated. Moreover, a pre-filtering algorithm was developed to classify good-quality and bad-quality spectra prior to the database searches. Th-lymphocytes can differentiate into Th1 or Th2 cells based on surrounding antigens, co-stimulatory molecules, and cytokines. Both subsets have individual cytokine secretion profiles and specific functions. Th1 cells participate in the cellular immunity against intracellular pathogens, while Th2 cells have important role in the humoral immunity against extracellular parasites. An abnormal response of Th1 and Th2 cells and imbalance between the subsets are charasteristic of several diseases. Th1 specific reactions and cytokines have been detected in autoimmune diseases, while Th2 specific response and cytokine profile is common in allergy and asthma. In this Ph. D. thesis mass spectrometry-based proteomics was used to study the effects of Th1 and Th2 promoting cytokines IL-12 and IL-4 on the proteome of Th lymphocytes. Characterization of microsomal fraction proteome extracted from IL-12 treated lymphobasts and IL-4 stimulated cord blood CD4+ cells resulted in finding of cytokine regulated proteins. Galectin-1 and CD7 were down-regulated in IL-12 treated cells, while IL-4 stimulation decreased the expression of STAT1, MXA, GIMAP1, and GIMAP4. Interestingly, the transcription of both GIMAP genes was up-regulated in Th1 polarized cells and down-regulated in Th2 promoting conditions.

The Confidence Information Ontology: a step towards a standard for asserting confidence in annotations.

Biocuration has become a cornerstone for analyses in biology, and to meet needs, the amount of annotations has considerably grown in recent years. However, the reliability of these annotations varies; it has thus become necessary to be able to assess the confidence in annotations. Although several resources already provide confidence information about the annotations that they produce, a standard way of providing such information has yet to be defined. This lack of standardization undermines the propagation of knowledge across resources, as well as the credibility of results from high-throughput analyses. Seeded at a workshop during the Biocuration 2012 conference, a working group has been created to address this problem. We present here the elements that were identified as essential for assessing confidence in annotations, as well as a draft ontology--the Confidence Information Ontology--to illustrate how the problems identified could be addressed. We hope that this effort will provide a home for discussing this major issue among the biocuration community. Tracker URL: https://github.com/BgeeDB/confidence-information-ontology Ontology URL: https://raw.githubusercontent.com/BgeeDB/confidence-information-ontology/master/src/ontology/cio-simple.obo

Tuotannon kehittäminen elektroniikan sopimusvalmistajayrityksessä

Tämän diplomityön tavoitteena on perehtyä Lean-ajatteluun ja osallistua kohdeyrityksen tuotannon kehittämiseen Lean-menetelmiä hyödyntäen. Tuotannon kehittämistoimenpiteiden tavoitteena on tuotannon läpäisyaikojen lyhentäminen, parempi kapasiteetin hallinta ja korkeampi tuottavuus. Keskeisimpiä työssä sovellettavia teorioita ovat Lean-valmistus, JIT ja jatkuva parantaminen. Työssä dokumentoidaan toteutetut kehittämistoimenpiteet sekä kartoitetaan kehitetyn tuotantoprosessin ongelmat. Saatujen tulosten perusteella pohditaan jatkotoimenpide-ehdotuksia ongelmien poistamiseksi. Työn tulokset osoittavat, että suurin haaste Lean-menetelmän käyttöönotossa on uusien ongelmien määrä, jotka paljastuvat vasta, kun prosessia kehitetään.

A highly expressed miR-101 isomiR is a functional silencing small RNA

Background MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression usually producing translational repression and gene silencing. High-throughput sequencing technologies have revealed heterogeneity at length and sequence level for the majority of mature miRNAs (IsomiRs). Most isomiRs can be explained by variability in either Dicer1 or Drosha cleavage during miRNA biogenesis at 5" or 3" of the miRNA (trimming variants). Although isomiRs have been described in different tissues and organisms, their functional validation as modulators of gene expression remains elusive. Here we have characterized the expression and function of a highly abundant miR-101 5"-trimming variant (5"-isomiR-101). Results The analysis of small RNA sequencing data in several human tissues and cell lines indicates that 5"-isomiR-101 is ubiquitously detected and a highly abundant, especially in the brain. 5"- isomiR-101 was found in Ago-2 immunocomplexes and complementary approaches showed that 5"-isomiR-101 interacted with different members of the silencing (RISC) complex. In addition, 5"-isomiR-101 decreased the expression of five validated miR-101 targets, suggesting that it is a functional variant. Both the binding to RISC members and the degree of silencing were less efficient for 5"-isomiR-101 compared with miR-101. For some targets, both miR-101 and 5"-isomiR-101 significantly decreased protein expression with no changes in the respective mRNA levels. Although a high number of overlapping predicted targets suggest similar targeted biological pathways, a correlation analysis of the expression profiles of miR-101 variants and predicted mRNA targets in human brains at different ages, suggest specific functions for miR-101- and 5"-isomiR-101. Conclusions These results suggest that isomiRs are functional variants and further indicate that for a given miRNA, the different isomiRs may contribute to the overall effect as quantitative and qualitative fine-tuners of gene expression.