Pharmacogenomics, 2004 Dec, 5(8), 1049 - 105 Genomic characterization of Alzheimer's disease and genotype-related phenotypic analysis of biological markers in dementia; Cacabelos R; More than 180 genes distributed across the human genome are potentially involved in the pathogenesis of Alzheimer's disease (AD) . The AD population shows a higher genetic variation rate than the control population . Significant differences in allelic distribution and frequency exist when AD-related polygenic clusters are compared with other forms of dementia, indicating that the genetic component in neurodegenerative dementia differs from that of other CNS disorders . The characterization of AD genotype-related phenotypic profiles reveals substantial differences in biological markers among AD clusters associated with different genes and/or allelic combinations . AD and dementia with vascular component (DVC) are the most prevalent forms of dementia . Both clinical entities share many similarities, but they differ in their major phenotypic and genotypic profiles, as revealed by structural and functional genomics studies . Comparative phenotypic studies have identified significant differences in 25% of more than 100 parametric variables, including anthropometric values, cardiovascular function, blood pressure, lipid metabolism, uric acid metabolism, peripheral calcium homeostasis, liver function, alkaline phosphatase, lactate dehydrogenase, red and white blood cells, regional brain atrophy, and brain blood flow velocity . Functional genomic studies incorporating apolipoprotein E (APOE)-related changes in biological markers extended the difference between AD and DVC by up to 57% . Structural genomic studies with AD-related genes, including APP, MAPT, APOE, PS1, PS2, A2M, ACE, AGT, cFOS, and PRNP, demonstrate different genetic profiles in AD and DVC, with an absolute genetic variation rate in the range of 30-80%, depending upon genes and genetic clusters . The relative polymorphic variation in genetic clusters integrated by two, three or four genes associated with AD ranges from 1 to 3% . The main phenotypic differences in AD are genotype dependent, indicating a powerful influence of polygenic factors on the AD phenotypic profile . All these genotypic and phenotypic variations bring about important consequences for the pharmacogenomics of AD. Expert Opin Ther Targets, 2004 Dec, 8(6), 653 - 61 Chemical genetics for therapeutic target mining; Shim JS et al.; Chemical genetics is an emerging research field that utilises biologically active small molecules to study biological functions of genes and their products . The direct regulation of the protein function by the biologically active small molecules can alternate the gene mutagenesis studies utilised in conventional genetics . Like conventional genetics, chemical genetics can be divided into two concepts - 'forward' and 'reverse' chemical genetics . These approaches of chemical genetics have a tremendous impact on both functional genomics and drug development . This review focuses on the two ways in which chemical genetics can be used for therapeutic target mining and their practical application in drug development, particularly, in angiogenesis-related diseases. Stem Cells, 2004, 22(7), 1330 - 7 Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord; Wang HS et al.; The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells . Using flow cytometric analysis, we found that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45) . Interestingly, these cells also express significant amounts of mesenchymal stem cell markers (SH2, SH3) . We therefore investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or by culturing them in cardiomyocyte-conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I . We also showed that these cells have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages . These findings may have a significant impact on studies of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical and technical issues. Curr Med Chem, 2004 Dec, 11(23), 3119 - 45 Recent advances in chemical genomics; Darvas F et al.; Chemical genomics, which utilizes specially designed small chemical compounds early in the discovery phase of new drugs to explore the life science at various levels, can address biological questions that are not amenable to genetic manipulation or functional genomics/proteomics approaches . Following the development of HT phenotypic assays and DNA expression analysis, the integration of cell-based assays with activity / affinity-based approaches allows us to interrogate the cells by analyzing phenotypic alterations, changes of transcript signature or detecting the differences in protein expression levels . Furthermore, activity / affinity-based techniques directly provide a druggable subset of gene products, which interact with small molecules, greatly reducing the complexity of analyzing the proteome . In this paper, we give an account of the recent advances (approaches and strategies) in the field of chemical genomics, and discuss how these approaches enable the investigator to obtain a novel therapeutically relevant target as well as drug candidates acting on them in a target-specific manner . This novel post-genomic discovery strategy, where target identification/ validation is carried out by interactions with small molecules, could significantly reduce the time-scale for early drug discovery, and increase the success rate of finding novel, druggable targets, as well as more specific drug candidates. Nucleic Acids Res . 2004 Dec;32(21):e171. RNA silencing in plants by the expression of siRNA duplexes; Lu S et al.; In animal cells, stable RNA silencing can be achieved by vector-based small interfering RNA (siRNA) expression system, in which Pol III RNA gene promoters are used to drive the expression of short hairpin RNA, however, this has not been demonstrated in plants . Whether Pol III RNA gene promoter is capable of driving siRNA expression in plants is unknown . Here, we report that RNA silencing was achieved in plants through stable expression of short hairpin RNA, which was driven by Pol III RNA gene promoters . Using glucuronidase (GUS) transformed tobacco as a model system, the results demonstrated that 21 nt RNA duplexes, targeting at different sites of GUS gene, were stably expressed under the control of either human H1 or Arabidopsis 7SL RNA gene promoter, and GUS gene was silenced in 80% of siRNA transgenics . The severity of silencing was correlated with the abundance of siRNA expression but independent of the target sites and uridine residue structures in siRNA hairpin transcripts . Thus, the specific expression of siRNA provides a new system for the study of siRNA silencing pathways and functional genomics in plants . Moreover, the effectiveness of the human H1 promoter in a plant background suggested a conserved mechanism underlying Pol III complex functionality. Science, 2004 Nov 26, 306(5701), 1555 - 8 A probabilistic functional network of yeast genes; Lee I et al.; A conceptual framework for integrating diverse functional genomics data was developed by reinterpreting experiments to provide numerical likelihoods that genes are functionally linked . This allows direct comparison and integration of different classes of data . The resulting probabilistic gene network estimates the functional coupling between genes . Within this framework, we reconstructed an extensive, high-quality functional gene network for Saccharomyces cerevisiae, consisting of 4681 (approximately 81%) of the known yeast genes linked by approximately 34,000 probabilistic linkages comparable in accuracy to small-scale interaction assays . The integrated linkages distinguish true from false-positive interactions in earlier data sets; new interactions emerge from genes' network contexts, as shown for genes in chromatin modification and ribosome biogenesis. Br J Haematol, 2004 Dec, 127(5), 479 - 90 Genetic polymorphisms predicting the outcome of bone marrow transplants; Dickinson AM et al.; Analysis of non-histocompatibility leucocyte antigen (HLA) functional genomics, together with conventional risk factors in haematopoietic stem cell transplantation (HSCT) can lead to predicting outcome in HLA-matched sibling transplant recipients . Polymorphisms of cytokine genes including tumour necrosis factor alpha, interleukin-10, interferon gamma and interleukin (IL)-6, associate with more severe acute graft-versus-host disease (aGvHD) . Donor genotype for IL-1 receptor antagonist (IL-1Ra) has been associated with reduced aGvHD severity . Other genotypes (patient IL-1Ra, IL-6 and donor IL-1 alpha) have been associated with chronic GvHD, or overall survival (Vitamin D receptor and oestrogen receptor) . Polymorphisms within genes associated with host defence/inflammatory responses (mannose binding lectin genes, myeloperoxidase genes and the FC gamma receptors) have been associated with infections . Polymorphisms of pharmacogenes, such as methylenetetrahydrofolate-reductase, have been associated with aGvHD and other post-transplant complications . The NOD2 gene polymorphism, associated with Crohn's disease, has been shown to be associated with risk of gut GvHD . The majority of the studies have been carried out in single centre HLA-matched sibling cohorts and in relatively few matched unrelated donor transplants . This review gives an overall perspective of the current field of non-HLA genetics with regard to HSCT outcome, clinical relevance and potential application of the results to clinical management of HSCT. Nat Genet, 2004 Dec, 36(12), 1306 - 11 Epub 2004 Dec. Application of comparative functional genomics to identify best-fit mouse models to study human cancer; Lee JS et al.; Genetically modified mice have been extensively used for analyzing the molecular events that occur during tumor development . In many, if not all, cases, however, it is uncertain to what extent the mouse models reproduce features observed in the corresponding human conditions . This is due largely to lack of precise methods for direct and comprehensive comparison at the molecular level of the mouse and human tumors . Here we use global gene expression patterns of 68 hepatocellular carcinomas (HCCs) from seven different mouse models and 91 human HCCs from predefined subclasses to obtain direct comparison of the molecular features of mouse and human HCCs . Gene expression patterns in HCCs from Myc, E2f1 and Myc E2f1 transgenic mice were most similar to those of the better survival group of human HCCs, whereas the expression patterns in HCCs from Myc Tgfa transgenic mice and in diethylnitrosamine-induced mouse HCCs were most similar to those of the poorer survival group of human HCCs . Gene expression patterns in HCCs from Acox1(-/-) mice and in ciprofibrate-induced HCCs were least similar to those observed in human HCCs . We conclude that our approach can effectively identify appropriate mouse models to study human cancers. Korean J Gastroenterol, 2004 Nov, 44(5), 239 - 45 {Proteomic approach in gastrointestinal and liver research}; Yeo M et al.; In the post-genomic era, the focus of research is now moving to functional genomics employing the information on predicted gene products provided by genome sequencing . Proteomics, the global analysis of structures, functions, and interactions of whole cellular proteins, draws the special attention as a tool for documenting the disease pathogenesis or progression . The high-throughput technology has become feasible by considerable improvement of two dimensional electrophoresis and mass fingerprinting . Thus proteome techniques can be used as tools to study the disease processes, develop new biomakers for diagnosis and early detection of diseases, and accelerate drug development . In this review, we discuss the background and techniques of proteomics, and potential applications to the research of gastrointestinal diseases. Cell Biol Int, 2004, 28(11), 755 - 64 Cell biology, chemogenomics and chemoproteomics; Gagna CE et al.; The scientific techniques used in molecular biological research and drug discovery have changed dramatically over the past 10 years due to the influence of genomics, proteomics and bioinformatics . Furthermore, genomics and functional genomics are now merging into a new scientific approach called chemogenomics . Advancements in the study of molecular cell biology are dependent upon "omics" researchers realizing the importance of and using the experimental tools currently available to cell biologists . For example, novel microscopic techniques utilizing advanced computer imaging allow for the examination of live specimens in a fourth dimension, viz., time . Yet, molecular biologists have not taken full advantage of these and other traditional and novel cell biology techniques for the further advancement of genomic and proteomic-oriented research . The application of traditional and novel cellular biological techniques will enhance the science of genomics . The authors hypothesize that a stronger interdisciplinary approach must be taken between cell biology (and its closely related fields) and genomics, proteomics and bio-chemoinformatics . Since there is a lot of confusion regarding many of the "omics" definitions, this article also clarifies some of the basic terminology used in genomics, and related fields . It also reviews the current status and future potential of chemogenomics and its relationship to cell biology . The authors also discuss and expand upon the differences between chemogenomics and the relatively new term--chemoproteomics . We conclude that the advances in cell biology methods and approaches and their adoption by "omics" researchers will allow scientists to maximize our knowledge about life. IDrugs, 2002 Mar, 5(3), 216 - 9 Proteolytic enzymes as therapeutic targets - keystone symposium; Creemers J; The Keystone Symposium 'Proteolytic Enzymes as Therapeutic Targets' was attended by approximately 150 scientists . Around two-thirds of the participants consisted of representatives from pharmaceutical companies, but representatives from academic institutes dominated the list of speakers . The meeting attracted scientists from many different fields, including biochemistry, molecular biology, structural biology, pharmacology, chemistry, and bioinformatics . The science ranged from the discovery and characterization of novel proteinases to the development and clinical trials of proteinase inhibitors and was presented as posters or in oral sessions . The discussions following the oral presentations were always very animated, but hardly ever heated . Although there were a few new drugs being presented, the real highlight was the enormous potential of recently discovered proteinases as new therapeutic targets . Both pharmaceutical companies and academic institutes are investing in programs to integrate the avalanche of new information coming from functional genomics, proteomics and structural information to create a platform for applied proteinase technology. Semin Cell Dev Biol, 2004 Dec, 15(6), 721 - 31 Bioinformatics: harvesting information for plant and crop science; King GJ; Bioinformatics is an integral aspect of plant and crop science research . Developments in data management and analytical software are reviewed with an emphasis on applications in functional genomics . This includes information resources for Arabidopsis and crop species, and tools available for analysis and visualisation of comparative genomic data . Approaches used to explore relationships between plant genes and expressed sequences are compared, including use of ontologies . The impact of bioinformatics in forward and reverse genetics is described, together with the potential from data mining . The role of bioinformatics is explored in the wider context of plant and crop science. Semin Cell Dev Biol, 2004 Dec, 15(6), 693 - 701 Integrative bioinformatics for functional genome annotation: trawling for G protein-coupled receptors; Flower DR et al.; G protein-coupled receptors (GPCR) are amongst the best studied and most functionally diverse types of cell-surface protein . The importance of GPCRs as mediates or cell function and organismal developmental underlies their involvement in key physiological roles and their prominence as targets for pharmacological therapeutics . In this review, we highlight the requirement for integrated protocols which underline the different perspectives offered by different sequence analysis methods . BLAST and FastA offer broad brush strokes . Motif-based search methods add the fine detail . Structural modelling offers another perspective which allows us to elucidate the physicochemical properties that underlie ligand binding . Together, these different views provide a more informative and a more detailed picture of GPCR structure and function . Many GPCRs remain orphan receptors with no identified ligand, yet as computer-driven functional genomics starts to elaborate their functions, a new understanding of their roles in cell and developmental biology will follow. Anal Biochem, 2004 Dec 15, 335(2), 260 - 6 High-quality RNA preparation for transcript profiling of osteocytes from native human bone microdissections; Eisenberger S et al.; Osteocytes, the most abundant bone cell type with important roles in tissue maintenance and pathological aberrations such as observed in bone metastases, are enclosed within a highly compact, calcified extracellular matrix . This location complicates analysis in native bone, with the consequence that despite their importance their in vivo molecular physiology is only poorly understood . We have examined the possibility of isolating osteocyte RNA for transcript profiling from native, frozen bone instead of employing the formalin-fixed, paraffin-embedded, decalcified version routinely used in histology, providing chemically modified and highly disintegrated RNAs . Bone tissue was tape-assisted cryosectioned and fixed to glass slides by support of UV-flash-triggered adhesive polymerization followed by quick hematoxylin-eosin staining to generate a guidance image for microdissection . Using an UVa-nitrogen laser, matrix-enclosed osteocytes were either excised and catapulted into RNA preparation vials or freed of accompanying nonosteocyte cellular material . The influences of bone sectioning, staining, and osteocyte capturing procedures on the prepared osteocyte RNAs were analyzed and the method was optimized accordingly . The obtained osteocyte RNAs showed the expected expression pattern of marker genes (reverse transcriptase-polymerase chain reaction), and, following conversion into fluorescent-labeled cDNAs, led to transcript profiles (cDNAchips; 2600 genes) with scatter-graph geometries indicating suitability for high-confidence evaluation . With the approach described here we introduce a methodological way for the characterization of the in vivo molecular physiology of osteocytes by functional genomics. Curr Opin Chem Biol, 2004 Dec, 8(6), 570 - 9 RNA interference and chemically modified small interfering RNAs; Manoharan M; RNA interference (RNAi) is a powerful biological process for specific silencing of gene expression in diversified eukaryotic cells and has tremendous potential for functional genomics, drug discovery through in vivo target validation, and development of novel gene-specific medicine . The future success of this technology relies on identifying appropriate chemical modifications to improve stability, potency and in vivo cellular delivery . The present review summarizes the role of the chemist's toolbox in this emerging technology. J Transl Med . 2004 Nov 22;2(1):39. RNA interference: learning gene knock-down from cell physiology; Mocellin S et al.; SUMMARY: Over the past decade RNA interference (RNAi) has emerged as a natural mechanism for silencing gene expression . This ancient cellular antiviral response can be exploited to allow specific inhibition of the function of any chosen target gene . RNAi is proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes . More importantly, RNAi technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes.This review briefly describes the molecular principles underlying the biology of RNAi phenomenon and discuss the main technical issues regarding optimization of RNAi experimental design. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Oct, 21(5), 848 - 51 {RNA interference in functional genomics and medical research}; You Z et al.; RNA interference (RNAi) is a post-transcriptional gene silencing process by targeting mRNA for degradation in a sequence-specific manner . This powerful platform has enormous potential in functional genomics and medical research . As a tool to knock out expression of specific genes in a variety of organisms, RNAi was used to investigate gene function in a high throughput fashion . Highly conserved in evolution RNAi appears to have evolved as a cellular defense mechanism in plants and animals to suppress viral infection, transposon jumping and endogenous aberrant genes . Exploiting the natural mechanism, the researchers can shut down disease-causing genes and develop novel therapeutics against infection, tumor and other disease. Physiol Genomics, 2004 Nov 17, 19(3), 233 - 46 Functional genomics of the dopaminergic system in hypertension; Zeng C et al.; Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension . Under normal conditions, D(1)-like receptors (D(1) and D(5)) inhibit sodium transport in the kidney and intestine . However, in the Dahl salt-sensitive and spontaneously hypertensive rats (SHRs) and in humans with essential hypertension, the D(1)-like receptor-mediated inhibition of epithelial sodium transport is impaired because of an uncoupling of the D(1)-like receptor from its G protein/effector complex . The uncoupling is receptor specific, organ selective, nephron-segment specific, precedes the onset of hypertension, and cosegregates with the hypertensive phenotype . The defective transduction of the renal dopaminergic signal is caused by activating variants of G protein-coupled receptor kinase type 4 (GRK4: R65L, A142V, A486V) . The GRK4 locus is linked to and GRK4 gene variants are associated with human essential hypertension, especially in salt-sensitive hypertensive subjects . Indeed, the presence of three or more GRK4 variants impairs the natriuretic response to dopaminergic stimulation in humans . In genetically hypertensive rats, renal inhibition of GRK4 expression ameliorates the hypertension . In mice, overexpression of GRK4 variants causes hypertension either with or without salt sensitivity according to the variant . GRK4 gene variants, by preventing the natriuretic function of the dopaminergic system and by allowing the antinatriuretic factors (e.g., angiotensin II type 1 receptor) to predominate, may be responsible for salt sensitivity . Subclasses of hypertension may occur because of additional perturbations caused by variants of other genes, the quantitative interaction of which may vary depending upon the genetic background. Autoimmun Rev, 2004 Nov, 3(7-8), 541 - 9 Strategies using functional genomics in rheumatic diseases; Burmester GR et al.; For functional genomics of inflammatory disorders and infection, rheumatic diseases offer unique features to analyse the transition from infection to chronic inflammation, autoimmunity and immunopathology, both systemic and tissue specific . The diseases are frequent and of considerable socio-economic impact . Well-defined cohorts of patients are available . The tissues and cells involved are readily accessible for molecular analysis . Both genetic predisposition and infection are involved in the aetiopathogenesis of rheumatic diseases . The number of susceptibility and severity genes has been estimated to be at least 30, but only few of them have been identified so far . There is an urgent need for developing new therapies adapted to genetic risk and based on a functional genetic and molecular understanding of chronic inflammation . It is evident that gene analysis in inflammatory rheumatic diseases will not only be beneficial for the large number of patients involved, but will also lead to a better understanding of other inflammatory disorders, thereby possibly leading to novel diagnostic and therapeutic strategies in this important group of disorders. Neurotox Res, 2004, 6(5), 363 - 72 Functional genomics strategies to identify susceptibility genes and treatment targets in alcohol dependence; Heilig M et al.; Genetic factors contribute to alcohol dependence through two main categories of mechanisms . The 50-60% heritability observed in this disorder is presumably conferred by polymorphic variants, encoding functionally altered proteins, or leading to differential transcriptional activity . Secondly, long term changes during the process of developing dependence are likely encoded by persistent changes in gene expression . Thus, genetic and environmental factors interact at the level of the transcriptome, making this an attractive level of analysis . For this purpose, we have applied differential display and more recently Affymetrix oligonucleotide gene arrays to models of genetic susceptibility and alcohol-induced neuroadaptation. Mol Biotechnol, 2004 Nov, 28(3), 195 - 9 Knockdown of mouse adult beta-globin gene expression in MEL cells by retrovirus vector-mediated RNA interference; Zhao N et al.; RNA interference (RNAi) efficiently induces sequence-specific gene silencing in mammalian cells through short interfering RNA (siRNA) of 21-23 nucleotides synthesized in vitro or expressed by DNA-based vector . However, introduction of siRNA into mammalian cells by transfection limits the application of RNAi, especially when it is necessary to generate long-term gene silencing in vivo . Virus vector-mediated RNAi provides an alternative to transfection . In the present study, we investigated such transduction system and showed that retrovirus vector-mediated RNAi can substantially down-regulate expression of mouse adult beta-globin gene in MEL cells . The results suggest that retrovirus vector-delivered RNAi may find its use in functional genomics and in gene therapy. Mol Cell Biol, 2004 Dec, 24(23), 10263 - 76 Multiple, distant Gata2 enhancers specify temporally and tissue-specific patterning in the developing urogenital system; Khandekar M et al.; Transcription factor GATA-2 is expressed in a complex temporally and tissue-specific pattern within the developing embryo . Loss-of-function studies in the mouse showed that GATA-2 activity is first required during very early hematopoiesis . We subsequently showed that a 271-kbp yeast artificial chromosome (YAC) transgene could fully complement the loss of Gata2 hematopoietic function but that these YAC-rescued Gata2 null mutant mice die perinatally due to defective urogenital development . The rescuing YAC did not display appropriate urogenital expression of Gata2, implying the existence of a urogenital-specific enhancer(s) lying outside the boundaries of this transgene . Here we outline a coupled general strategy for regulatory sequence discovery, linking bioinformatics to functional genomics based on the bacterial artificial chromosome (BAC) libraries used to generate the mouse genome sequence . Exploiting this strategy, we screened >1 Mbp of genomic DNA surrounding Gata2 for urogenital enhancer activity . We found that the spatially and tissue-specific functions for Gata2 in the developing urogenital system are conferred by at least three separate regionally and temporally specific urogenital enhancer elements, two of which reside far 3' to the Gata2 structural gene . Including the additional enhancers that were discovered using this strategy (called BAC trap) extends the functional realm of the Gata2 locus to greater than 1 Mbp. Plant Physiol, 2004 Dec, 136(4), 4228 - 36 Epub 2004 Nov 12. Rice contains two disparate ent-copalyl diphosphate synthases with distinct metabolic functions; Prisic S et al.; Rice (Oryza sativa) produces ent-copalyl diphosphate for both gibberellin (GA) phytohormone and defensive phytoalexin biosynthesis, raising the question of how this initial biosynthetic step is carried out for these distinct metabolic processes . Here, a functional genomics approach has been utilized to identify two disparate ent-copalyl diphosphate synthases from rice (OsCPS1ent and OsCPS2ent) . Notably, it was very recently demonstrated that only one of these (OsCPS1ent) normally operates in GA biosynthesis as mutations in this gene result in severely impaired growth . Evidence is presented here strongly indicating that the other (OsCPS2ent) is involved in related secondary metabolism producing defensive phytochemicals . In particular, under appropriate conditions, OsCPS2ent mRNA is specifically induced in leaves prior to production of the corresponding phytoalexins . Thus, transcriptional control of OsCPS2ent seems to be an important means of regulating defensive phytochemical biosynthesis . Finally, OsCPS1ent is significantly more similar to the likewise GA-specific gene An1/ZmCPS1ent in maize (Zea mays) than its class II terpene synthase paralogs involved in rice secondary metabolism . Hence, we speculate that this cross-species conservation by biosynthetic process reflects derivation of related secondary metabolism from the GA primary biosynthetic pathway prior to the early divergence between the separate lineages within the cereal/grass family (Poaceae) resulting in modern rice and maize. Cancer Biol Ther . 2004 Nov 12;3(11) {Epub ahead of print} siRNAs: Mechanism of RNA Interference, In Vivo and Potential Clinical Applications; Karagiannis TC et al.; Small interfering RNAs are currently the most widely used nucleic acid-based sequence-specific gene silencing molecules . These molecules mediate RNA interference-a natural post-transcriptional gene-silencing pathway . Given the high reliability and higher efficiency of small interfering RNA-mediated RNA interference, compared to earlier reverse genetic technologies, this is now the preferred technique in functional genomics . Furthermore, the exquisite specificity and exceptional gene-silencing potency of small interfering RNAs has resulted in intense research related to potential target-specific therapeutic applications of these molecules . This review will discuss the mechanism of RNA interference and applications of the pathway in molecular biology including functional genomics will be overviewed . The article will outline in vivo and potential clinical applications of small interfering RNA molecules. Anal Chem, 2004 Nov 15, 76(22), 6555 - 9 Surface plasmon resonance-based sensors to identify cis-regulatory elements; Lin L et al.; In eukaryotes, transcription is regulated by multiprotein complexes binding to specific regions of genomic DNA, called cis-regulatory elements . Comprehensive identification of these elements is an important goal of functional genomics . Hence, it is of practical interest to develop a high-throughput assay to identify cis-regulatory elements . Toward that goal, we demonstrate that a surface plasmon resonance-based assay can identify whether a specific region of DNA binds to proteins present in raw nuclear lysate . Specifically, we immobilized a 16-basepair double-stranded DNA region of the SQSTM1 promoter to the Texas Instruments Spreeta, a surface plasmon resonance sensor . As a control, in a separate experiment, we immobilized a similar piece of DNA that differed by only a single base pair . We observed a significant difference in surface plasmon resonance signal when these two probes were exposed to raw nuclear lysate from NIH/3T3 cells . Using a luciferase-reporter vector transfected into live NIH/3T3 cells, we measured a significant difference in transcriptional activity between the two pieces of DNA . We conclude that a surface plasmon resonance-based sensor is capable of identifying physiologically significant cis-regulatory elements. Microb Cell Fact . 2004 Nov 10;3(1):13. The bag or the spindle: the cell factory at the time of systems' biology; Danchin A; Genome programs changed our view of bacteria as cell factories, by making them amenable to systematic rational improvement . As a first step, isolated genes (including those of the metagenome), or small gene clusters are improved and expressed in a variety of hosts . New techniques derived from functional genomics (transcriptome, proteome and metabolome studies) now allow users to shift from this single-gene approach to a more integrated view of the cell, where it is more and more considered as a factory . One can expect in the near future that bacteria will be entirely reprogrammed, and perhaps even created de novo from bits and pieces, to constitute man-made cell factories . This will require exploration of the landscape made of neighbourhoods of all the genes in the cell . Present work is already paving the way for that futuristic view of bacteria in industry. Neuroendocrinology, 2004, 80(2), 92 - 9 Epub 2004. Vasopressin stimulates ventromedial hypothalamic neurons via oxytocin receptors in oxytocin gene knockout male and female mice; Ragnauth AK et al.; A wealth of neuropharmacological data demonstrates that oxytocin (OT) actions in the mammalian forebrain support a wide variety of affiliative behaviors and repress aggressive behaviors . Based on that literature, it was expected that reproductive and affiliative behaviors would be vastly decreased and aggression markedly increased in OT gene knockout (OTKO) mice . The initial publications reporting the behaviors of these mice did not include such phenotypes . Here, we compared single-unit activities recorded from the ventromedial hypothalamus in tissue slices of male and female OTKO mice and their wild-type littermate to test two hypotheses about OT functional genomics . First, we proposed that in OTKO mice, a very similar 9-amino-acid neuropeptide, arginine vasopressin (a likely gene duplication product), can 'cross over' and compensate for the lack of OT . This hypothesis was confirmed in both males and females . Further, we proposed that because of the lifelong absence of OT in OTKO, OT receptors would be more sensitive to OT in the knockout animals . We tested this idea in males and found that it was correct . Thus, an answer to the 'OTKO paradox' is put forth, with implications for OT-sensitive behaviors in a variety of species. J Am Acad Dermatol, 2004 Nov, 51(5), 681 - 92; quiz 693-6 DNA microarrays: from structural genomics to functional genomics . The applications of gene chips in dermatology and dermatopathology; Sellheyer K et al.; The human genome project was successful in sequencing the entire human genome and ended earlier than expected . The vast genetic information now available will have far-reaching consequences for medicine in the twenty-first century . The knowledge gained from the mapping and sequencing of human genes on a genome-wide scale--commonly referred to as structural genomics--is prerequisite for studies that focus on the functional aspects of genes . A recently invented technique, known as gene chip, or DNA microarray, technology, allows the study of the function of thousands of genes at once, thereby opening the door to the new field of functional genomics . At its core, the DNA microarray utilizes a unique feature of DNA known as complementary hybridization . As such, it is not different from Southern (DNA) blot or northern (RNA) blot hybridizations, or the polymerase chain reaction, with the exception that it allows expression profiling of the entire human genome in a single hybridization experiment . The article highlights the principles, technology, and applications of DNA microarrays as they pertain to the field of dermatology and dermatopathology . The most important applications are the gene expression profiling of skin cancer, especially of melanoma . Other potential applications include gene expression profiling of inflammatory skin diseases, the mutational analysis of genodermatoses, and polymorphism screening, as well as drug development and chemosensitivity prediction . cDNA microarrays will shape the diagnostic approach of the dermatology and the dermatopathology of the future and may lead to new therapeutic options. J Gene Med . 2004 Nov 1; {Epub ahead of print} Engineered Streptomyces quorum-sensing components enable inducible siRNA-mediated translation control in mammalian cells and adjustable transcription control in mice; Weber W et al.; BACKGROUND: Recent advances in functional genomics, gene therapy, tissue engineering, drug discovery and biopharmaceuticals production have been fostered by precise small-molecule-mediated fine-tuning of desired transgenes . METHODS: Capitalizing on well-evolved quorum-sensing regulatory networks in Streptomyces coelicolor we have designed a mammalian regulation system inducible by the non-toxic butyrolactone SCB1 . Fusion of the S . coelicolor SCB1 quorum-sensing receptor ScbR to the human Kox-1-derived transsilencing domain reconstituted a mammalian transsilencer (SCS) able to repress transcription from SCS-specific operator-containing promoters in a reverse SCB1-adjustable manner . RESULTS: This quorum-sensing-derived mammalian transgene control system (Q-ON) enabled precise SCB1-specific fine-tuning of (i) desired transgene transcription in a variety of mammalian/human cell lines and human primary cells, (ii) small interfering RNA-mediated posttranscriptional knockdown (siRNA) in mammalian cells, and (iii) dosing of a human glycoprotein in mice . CONCLUSIONS: As exemplified by Q-ON technology, bacterial quorum-sensing regulons may represent a near-infinite source for the design of mammalian gene control systems compatible with molecular interventions relevant to future gene therapy and tissue engineering scenarios . Copyright (c) 2004 John Wiley & Sons, Ltd. Mamm Genome, 2004 Oct, 15(10), 768 - 83 Mouse functional genomics requires standardization of mouse handling and housing conditions; Champy MF et al.; The study of mouse models is crucial for the functional annotation of the human genome . The recent improvements in mouse genetics now moved the bottleneck in mouse functional genomics from the generation of mutant mice lines to the phenotypic analysis of these mice lines . Simple, validated, and reproducible phenotyping tests are a prerequisite to improving this phenotyping bottleneck . We analyzed here the impact of simple variations in animal handling and housing procedures, such as cage density, diet, gender, length of fasting, as well as site (retro-orbital vs . tail), timing, and anesthesia used during venipuncture, on biochemical, hematological, and metabolic/endocrine parameters in adult C57BL/6J mice . Our results, which show that minor changes in procedures can profoundly affect biological variables, underscore the importance of establishing uniform and validated animal procedures to improve reproducibility of mouse phenotypic data. Nat Rev Drug Discov, 2004 Nov, 3(11), 965 - 72 Functional genomics to new drug targets; Kramer R et al.; The completion of the sequencing of the human genome, and those of other organisms, is expected to lead to many potential new drug targets in various diseases, and it is predicted that novel therapeutic agents will be developed against such targets . The role of functional genomics in modern drug discovery is to prioritize these targets and to translate that knowledge into rational and reliable drug discovery . Here, we describe the field of functional genomics and review approaches that have been applied to drug discovery, including RNA profiling, proteomics, antisense and RNA interference, model organisms and high-throughput, genome-wide overexpression or knockdowns, and outline the future directions that are likely to yield new drug targets from genomics. J Exp Bot, 2005 Jan, 56(410), 255 - 65 Epub 2004 Nov 01. Metabolite fingerprinting and profiling in plants using NMR; Krishnan P et al.; Although less sensitive than mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy provides a powerful complementary technique for the identification and quantitative analysis of plant metabolites either in vivo or in tissue extracts . In one approach, metabolite fingerprinting, multivariate analysis of unassigned (1)H NMR spectra is used to compare the overall metabolic composition of wild-type, mutant, and transgenic plant material, and to assess the impact of stress conditions on the plant metabolome . Metabolite fingerprinting by NMR is a fast, convenient, and effective tool for discriminating between groups of related samples and it identifies the most important regions of the spectrum for further analysis . In a second approach, metabolite profiling, the (1)H NMR spectra of tissue extracts are assigned, a process that typically identifies 20-40 metabolites in an unfractionated extract . These profiles may also be used to compare groups of samples, and significant differences in metabolite concentrations provide the basis for hypotheses on the underlying causes for the observed segregation of the groups . Both approaches generate a metabolic phenotype for a plant, based on a system-wide but incomplete analysis of the plant metabolome . However, a review of the literature suggests that the emphasis so far has been on the accumulation of analytical data and sample classification, and that the potential of (1)H NMR spectroscopy as a tool for probing the operation of metabolic networks, or as a functional genomics tool for identifying gene function, is largely untapped. Oncogene, 2004 Nov 1, 23(51), 8401 - 9 RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age; Silva J et al.; Sequencing of complete genomes has provided researchers with a wealth of information to study genome organization, genetic instability, and polymorphisms, as well as a knowledge of all potentially expressed genes . The identification of all genes encoded in the human genome opens the door for large-scale systematic gene silencing using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) . With the recent development of siRNA and shRNA expression libraries, the application of RNAi technology to assign function to cancer genes and to delineate molecular pathways in which these genes affect in normal and transformed cells, will contribute significantly to the knowledge necessary to develop new and also improve existing cancer therapy. Oncogene, 2004 Nov 1, 23(51), 8384 - 91 Oncology studies using siRNA libraries: the dawn of RNAi-based genomics; Sachse C et al.; High-throughput, human cell-based applications of RNA-mediated interference (RNAi) have emerged in recent years as perhaps the most powerful of a 'second wave' of functional genomics technologies . The available reagents and methodologies for RNAi screening studies now enable a wide range of different scopes and scales of investigation, from single-parameter assays applied to focused subsets of genes, to comprehensive genome-wide surveys based on rich, multiparameter readouts . As such, RNAi-based screens are offering important new avenues for the discovery and validation of novel therapeutic targets for several disease areas, including oncology . By enabling a 'clean' determination of gene function, that is the creation of direct causal links between gene and phenotype in human cells, RNAi investigations promise levels of pathophysiological relevance, efficiency, and range of applicability never before possible on this scale . The field of oncology, with its many assays using readily transfectable cell lines, has offered particularly fertile ground for showcasing the potential of RNAi-based genomics . However, like any other technology before it, RNAi is not without its own challenges, limitations, and caveats . Many of these issues stem directly from the choice of silencing reagent to be used in such studies, and the design of the overall screening strategy . Here, we discuss the basic design issues, potential advantages, and technical challenges of large-scale RNAi screens based on the use of chemically synthesized siRNA libraries. Oncogene, 2004 Nov 1, 23(51), 8376 - 83 Use of RNA interference libraries to investigate oncogenic signalling in mammalian cells; Downward J; Over the past decade, 'RNA interference' has emerged as a natural mechanism of silencing of gene expression . This ancient cellular antiviral response can be manipulated to provide an effective research tool to knock down the level of expression of selected target genes, providing a very powerful new method for the analysis of cell signalling pathways . Systematic silencing of genes on a genome-wide scale using large rationally designed libraries targeting many thousands of genes provides a novel functional genomics approach to the investigation of many aspects of mammalian cell behaviour, including oncogenic transformation . Here, the different approaches taken to use RNA interference libraries to study the cancer phenotype will be considered, including both selective and high throughput screens and the use of both vector-based and synthetic oligonucleotide-based methods for inducing RNA interference . The advantages and drawbacks of the competing methodologies will be discussed . RNA interference library technology holds great promise for enabling somatic cell genetics in tissue culture systems . Whether it can provide significant new insights into cancer will be its greatest challenge. Oncogene, 2004 Nov 1, 23(51), 8359 - 65 Using RNAi to catch Drosophila genes in a web of interactions: insights into cancer research; Dasgupta R et al.; The completion of whole-genome sequencing of various model organisms and the recent explosion of new technologies in the field of Functional Genomics and Proteomics is poised to revolutionize the way scientists identify and characterize gene function . One of the most significant advances in recent years has been the application of RNA interference (RNAi) as a means of assaying gene function . In the post-genomic era, advances in the field of cancer biology will rely upon the rapid identification and characterization of genes that regulate cell growth, proliferation, and apoptosis . Significant efforts are being directed towards cancer therapy and devising efficient means of selectively delivering drugs to cancerous cells . In this review, we discuss the promise of integrating genome-wide RNAi screens with proteomic approaches and small-molecule chemical genetic screens, towards improving our ability to understand and treat cancer. Oncogene, 2004 Nov 1, 23(51), 8353 - 8 High-throughput gene silencing using cell arrays; Vanhecke D et al.; A recently established transfected cell array (TCA) technology has opened new experimental dimensions in the field of functional genomics . Cell arrays allow for transfection of several thousands different DNA molecules in microarray format . The effects of overexpression of hundreds of proteins on cellular physiology can be observed in a single experiment . The TCA technique has also found its application in RNA interference (RNAi) research . Small interfering RNAs (siRNA) as well as plasmid expressing short hairpin RNAs can be transferred into the cells through the process of reverse transfection . The silencing of numerous genes in spatially separated manner can be thus monitored . This review will provide an overview on current concepts concerning combination of cell array and RNAi for high-throughput loss-of-function studies. J Nippon Med Sch, 2004 Oct, 71(5), 306 - 7 Thin is better!: ultrathin cryosection immunocytochemistry; Takizawa T et al.; In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images . One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope . The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm . Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections) . The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness . The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig . 1) . Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig . 2) . We show new methods of ultrathin cryosection immunocytochemistry(1-3) . Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen . Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM . Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles . For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol(1) . Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules(2,3). Biol Reprod . 2004 Oct 27; {Epub ahead of print} Human Myometrial Gene Expression Before and During Parturition; Havelock JC et al.; Identification of temporal and spatial changes in myometrial gene expression during parturition may further the understanding of the coordinated regulation of myometrial contractions during parturition . The objective of this study was to compare the gene expression profiles of human fundal myometrium from pregnant women before and after the onset of labor using a functional genomics approach, and to further characterize the spatial and temporal expression patterns of three genes thought to be important in parturition . Fundal myometrial mRNA was isolated from 5 women in labor and 5 women not in labor and analyzed using human UniGEM-V microarrays with 9182 cDNA elements . Real-time PCR using myometrial RNA from pregnant women in labor or not in labor was used to examine mRNA levels for three of the genes, namely prostaglandin-endoperoxide synthase 2 (PTGS2), calgranulin B (S100A9), and oxytocin receptor (OXTR) . The spatial expression pattern of these genes throughout the pregnant uterus before and after labor was also determined . Immunolocalization of COX-2 (also known as PTGS2) and S100A9 within the uterine cervix and myometrium were analyzed by immunohistochemistry . Few genes were differentially expressed in fundal myometrial tissues at term with the onset of labor . However, there appears to be a subset of genes important in the parturition cascade . The cellular properties of S100A9, its spatial localization, and dramatic increase in cervix and myometrium of laboring women suggest that this protein may be very important in the initiation or propagation of human labor. Am J Obstet Gynecol, 2004 Oct, 191(4), 1331 - 8 Genome-wide expression profiling of fetal membranes reveals a deficient expression of proteinase inhibitor 3 in premature rupture of membranes; Tromp G et al.; OBJECTIVE: We used a genome-wide approach to identify differentially expressed genes in patients with preterm premature rupture of membranes to improve the understanding of underlying molecular mechanisms . STUDY DESIGN: RNA was isolated from the fetal membranes of patients with preterm labor with intact membranes and preterm premature rupture of membranes and was stratified according to the presence or absence of histologic chorioamnionitis . Microarray experiments were used to identify differentially expressed genes, and real-time quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry were used in follow-up experiments . RESULTS: Microarray experiments identified decreased expression of proteinase inhibitor 3 in the preterm premature rupture of membranes cases . Quantitative reverse transcriptase-polymerase chain reaction confirmed these results . Immunohistochemistry demonstrated decreased proteinase inhibitor 3 protein expression in preterm premature rupture of membranes . CONCLUSION: A genome-wide approach identified deficient expression of proteinase inhibitor 3 in preterm premature rupture of membranes, which demonstrated the usefulness of functional genomics for the dissection of mechanisms of disease and identification of differentially regulated genes that were not suspected previously to play a role in parturition. J Virol, 2004 Nov, 78(22), 12576 - 90 Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis; Song WJ et al.; Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV) . Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp . One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified . Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins . Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database . An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified . Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation . The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands . Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins . Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products. Plant Cell Rep . 2004 Oct 19; {Epub ahead of print} A rapid and efficient transformation protocol for the grass Brachypodium distachyon; Christiansen P et al.; A fast and efficient microprojectile bombardment-mediated transformation protocol is reported for the grass species Brachypodium distachyon, a proposed alternative model plant to Oryza sativa for functional genomics in grasses . Embryogenic calli derived from immature embryos were transformed by a construct containing the uidA (coding for beta-glucuronidase) and bar (coding for phosphinothricin acetyl transferase) genes, and bialaphos, a non-selective herbicide, was used as the selection agent throughout all phases of the tissue culture . Average transformation efficiencies of 5.3% were achieved, and for single bombardments transformation efficiencies of up to 14% were observed . The time frame from the bombardment of embryogenic callus to the harvesting of transgenic T(1) seeds was 29 weeks and 25 weeks for the diploid and two tetraploid accessions used, respectively . Since the seed-to-seed life cycle is 19 weeks for the diploid and 15 weeks for the tetraploid accessions, our B . distachyon transformation system allows testing of both the T(0) and the T(1) generation as well as production of T(2) seeds within 1 year. Plant J, 2004 Nov, 40(4), 611 - 21 Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas; Rohr J et al.; RNA interference (RNAi), the double-stranded RNA (dsRNA) triggered post-transcriptional gene silencing, is becoming a powerful tool for reverse genetics studies . Stable RNAi, induced by the expression of inverted repeat (IR) transgenes, has been achieved in protozoa, algae, fungi, plants, and metazoans . However, the level of gene silencing is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene . This is a hindrance in functional genomics studies, where it is desirable to suppress target genes reliably to analyze unknown phenotypes . Consequently, we explored strategies for direct selection of effective transgenic RNAi lines in Chlamydomonas reinhardtii . We initially attempted to suppress expression of the Rubisco small subunit multigene family by placing an IR, homologous to the conserved coding sequence, in the 3'UTR of a transgene conferring resistance to bleomycin . However, this approach was fairly inefficient at inducing RNAi as many strains displayed defective transgene integration, resulting in partial or complete deletion of the IR, or low levels of dsRNA expression, presumably due to transcriptional silencing of the integrated IR transgenes . To overcome these problems we designed a system consisting of tandem IR transgenes that consistently triggered co-silencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta subunit) and another gene of interest (encoding either Ku80, an RNA-binding protein, or a thioredoxin isoform) . We anticipate that this approach will be useful for generating stable hypomorphic epi-mutants in high-throughput phenotypic screens. Wien Klin Wochenschr, 2002, 114 Suppl 4, 4 - 9 {Pathophysiology and genetics of obesity}; Oberkofler H et al.; Obesity has become the most prevalent nutritional disorder in post-industrialised societies and it is associated with the development of severe and costly complications such as type 2 diabetes mellitus and coronary heart disease or cancer . A large proportion of the risk of obesity is determined by the genetic susceptibility of an individual, but environmental factors conducive for the disorder play an important role in its phenotypic expression . Several candidate genes emerged from studies in animal models of obesity, but human pathophysiology is likely to be more complex . Thus, most cases of human obesity probably result from subtle interactions of susceptibility genes with environmental factors favouring deposition of excess calories as fat . The recent surge of obesity may relate to past evolutionary pressure which favoured selection of mechanisms defending body-weight against caloric restriction rather than against caloric excess . Rapidly developing new techniques in quantitative genetics and growing information from functional genomics will help to understand the interaction of environmental factors with signalling networks that regulate energy metabolism . The role of previously unknown pathways in the aetiology of obesity will be uncovered . The typing of numerous genetic variants will become possible and allow individual risk assessment for obesity and/or its associated disorders . Thus, rational and individually tailored therapies may be developed to combat obesity and its associated disorders. Methods Mol Med, 2004, 105, 439 - 52 A functional genomics approach to hematopoietic stem cell regulation; Hackney JA et al.; Elucidation of the molecular mechanisms that are responsible for regulating the most basic properties of stem cells, self-renewal, and differentiation remains a major challenge in hematopoietic stem cell biology . We have taken a functional genomics approach towards revealing these mechanisms . Previous studies of the fetal liver genetic program led to the development of Stem Cell Database, a resource for the stem cell community . These studies have been expanded to include the microenvironmental component of hematopoiesis and are the focus herein . In our efforts to study the microenvironmental component we have identified a stromal cell line, AFT024, which serves as a surrogate stem cell niche . The line provides a milieu that facilitates the maintenance of transplantable mouse and human stem cells as well as the generation of large populations of committed progenitors . In a manner mirroring the work done with the SCDb, we provide an online resource, Stromal Cell Database, StroCDB, that is a compendium of information and data derived from biological and molecular studies of this surrogate niche . These include bioinformatic analyses of over 6000 clones derived from a subtracted library enriched for messages expressed in AFT024 as well as data derived from custom expression arrays developed from this library . Herein we describe these efforts and provide a guide for navigating the database and mining the information contained within. Biochemistry, 2004 Oct 26, 43(42), 13348 - 56 Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA; Spagnou S et al.; RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells {Novina, C . D., and Sharp, P . A . (2004) The RNAi revolution, Nature 430, 161-164} . Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells . As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function . However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects . For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies . Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies . Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) . Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects . Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles . Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown . Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity . Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles . Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000 . Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection . By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles. Genome Res, 2004 Oct, 14(10B), 2176 - 89 Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications; Hilson P et al.; Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes . Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches . The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome . They were synthesized by PCR amplification from genomic DNA . Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments . The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols . These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics . We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells . We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines . These versatile GST resources provide novel and powerful tools for functional genomics. Genome Res, 2004 Oct, 14(10B), 2136 - 44 From ORFeome to biology: a functional genomics pipeline; Wiemann S et al.; As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease . We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction . Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network . We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information . All information is presented in a Web database . It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy. Genome Res, 2004 Oct, 14(10B), 2076 - 82 High-throughput generation of P . falciparum functional molecules by recombinational cloning; Aguiar JC et al.; Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression . We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes . Master clones and their ORFs were transferred en masse to multiple expression vectors . Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure . Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency . The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors . These transfers yielded a 100% success rate without any observed recombination based on single colony screening . The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite . From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages . We have overcome the potential limitation of producing large P . falciparum clone sets in multiple expression vectors . This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P . falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories. Annu Rev Genomics Hum Genet, 2004, 5, 443 - 77 Plant genomics: the third wave; Borevitz JO et al.; Completing the primary genomic sequence of Arabidopsis thaliana was a major milestone, being the first plant genome and only the third high-quality finished eukaryotic genome sequence . Understanding how the genome sequence comprehensively encodes developmental programs and environmental responses is the next major challenge for all plant genome projects . This requires fully characterizing the genes, the regulatory sequences, and their functions . We discuss several functional genomics approaches to decode the linear sequence of the reference plant Arabidopsis thaliana, including full-length cDNA collections, microarrays, natural variation, knockout collections, and comparative sequence analysis . Genomics provides the essential tools to speed the work of the traditional molecular geneticist and is now a scientific discipline in its own right. Annu Rev Genomics Hum Genet, 2004, 5, 267 - 93 Proteomics; de Hoog CL et al.; The genome sequences of important model systems are available and the focus is now shifting to large-scale experiments enabled by this data . Following in the footsteps of genomics, we have functional genomics, proteomics, and even metabolomics, roughly paralleling the biological hierarchy of the transcription, translation, and production of small molecules . Proteomics is initially concerned with determining the structure, expression, localization, biochemical activity, interactions, and cellular roles of as many proteins as possible . There has been great progress owing to novel instrumentation, experimental strategies, and bioinformatics methods . The area of protein-protein interactions has been especially fruitful . First pass interaction maps of some model organisms exist, and the proteins in many important organelles are about to be determined . Researchers are also beginning to integrate large-scale data sets from various "omics" disciplines in targeted investigations of specific biomedical areas and in pursuit of a general framework for systems biology. Clin Orthop, 2004 Oct, (427 Suppl), S138 - 43 Functional genomics of osteoarthritis: on the way to evaluate disease hypotheses; Aigner T et al.; Functional genomics is a challenging new way to address complex diseases such as osteoarthritis on a molecular level . This complements previous research and will open up new areas of so far unrecognized molecular networks . In this respect, articular cartilage is a good target for functional genomics as it contains only one cell type to which all expression signals can be attributed to . Despite considerable limitations at present, such as a low sensitivity and insensitivity to alternative splicing, posttranscriptional regulation, and posttranslational modification, cDNA-array technology provides a powerful tool to obtain an overview on gene expression patterns hardly achievable with other techniques . This has been shown to be true for known genes as well as for the identification of new genes of interest . Therefore, gene expression analysis will help to identify single genes depending on the disease and experimental conditions investigated . However, the expression pattern of the plethora of expressed genes will paint a picture (network) of disease context, maybe even more pushing forward our understanding of complex diseases such as osteoarthritis. FEMS Immunol Med Microbiol, 2004 Nov 1, 42(3), 271 - 9 Current concepts on Escherichia coli K1 translocation of the blood-brain barrier; Xie Y et al.; The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy . Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis . Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity . We have previously established both in vitro and in vivo models of the blood-brain barrier (BBB) using human brain microvascular endothelial cells (HBMEC) and hematogenous meningitis in neonatal rats, respectively . With these in vitro and in vivo models, we have shown that successful crossing of the BBB by circulating E . coli requires a high-degree of bacteremia, E . coli binding to and invasion of HBMEC, and E . coli traversal of the BBB as live bacteria . Our previous studies using TnphoA, signature-tagged mutagenesis and differential fluorescence induction identified several E . coli K1 determinants such as OmpA, Ibe proteins, AslA, TraJ and CNF1 contributing to invasion of HBMEC in vitro and traversal of the blood-brain barrier in vivo . We have shown that some of these determinants interact with specific receptors on HBMEC, suggesting E . coli translocation of the BBB is the result of specific pathogen-host cell interactions . Recent studies using functional genomics techniques have identified additional E . coli K1 factors that contribute to the high degree of bacteremia and HBMEC binding/invasion/transcytosis . In this review, we summarize the current knowledge on the mechanisms underlying the successful E . coli translocation of the BBB. Sci Aging Knowledge Environ . 2004 Oct 06;2004(40):pe37. Genes, longevity, and technology: meeting report from the 2nd conference on functional genomics of aging in Crete; Baumeister R et al.; Whole-genome sequences are now available, and methods have evolved for targeting, in parallel, each gene in a genome, offering for the first time the opportunity to study the entire dynamic network of genes involved in aging . At a recent conference in Hersonissos, Crete, around 200 internationally renowned experts gathered to discuss techniques and emerging results as the science of aging undergoes a shift toward systems biology. Curr Opin Biotechnol, 2004 Oct, 15(5), 444 - 8 Using functional genomics to improve productivity in the manufacture of industrial biochemicals; Hermann T; Recent developments in the field of functional genomics have been used to increase productivity in the manufacture of industrial biochemicals . Technologies like transcriptomics and proteomics have profited from the increasing number of genome sequencing projects . Meanwhile functional genomics has evolved from several isolated technologies, such as DNA chip technology and proteomics, to combined approaches that can help us to understand why organisms produce a certain product . The combination of expression studies and kinetic studies, such as carbon flux determination or metabolite measurements, has significantly improved productivity in production processes. Eur J Pharmacol, 2004 Oct 1, 500(1-3), 385 - 98 Proteomic approaches in brain research and neuropharmacology; Vercauteren FG et al.; Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions . Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents . Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein-protein interactions of thousands of proteins at a time . In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed . The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed . It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade. Int J Cancer, 2005 Jan 20, 113(3), 434 - 9 High throughput functional genomics: identification of novel genes with tumor suppressor phenotypes; Koenig-Hoffmann K et al.; We have used a combination of high throughput functional genomics, computerized database mining and expression analyses to discover novel human tumor suppressor genes (TSGs) . A genome-wide high throughput cDNA phenotype screen was established to identify genes that induce apoptosis or reduce cell viability . TSGs are expressed in normal tissue and frequently act by reduction of growth of transformed cells or induce apoptosis . In agreement with that and thus serving as platform validation, our pro-apoptotic hits included genes for which tumor suppressing activities were known, such as kangai1 and CD81 antigen . Additional genes that so far have been claimed as putative TSGs or associated with tumor inhibitory activities (prostate differentiation factor, hRAS-like suppressor 3, DPH2L1-like and the metastasis inhibitor Kiss1) were confirmed in their proposed TSG-like phenotype by functionally defining their growth inhibitory or pro-apoptotic function towards cancer cells . Finally, novel genes were identified for which neither association with cell growth nor with apoptosis were previously described . A subset of these genes show characteristics of TSGs because they (i) reduce the growth or induce apoptosis in tumor cells; (ii) show reduced expression in tumor vs . normal tissue; and (iii) are located on chromosomal (LOH-) loci for which cancer-associated deletions are described . The pro-apoptotic phenotype and differential expression of these genes in normal and malignant tissue make them promising target candidates for the diagnosis and therapy of various tumors. Biophys J, 2004 Oct, 87(4), 2647 - 55 Tertiary structure predictions on a comprehensive benchmark of medium to large size proteins; Zhang Y et al.; We evaluate tertiary structure predictions on medium to large size proteins by TASSER, a new algorithm that assembles protein structures through rearranging the rigid fragments from threading templates guided by a reduced Calpha and side-chain based potential consistent with threading based tertiary restraints . Predictions were generated for 745 proteins 201-300 residues in length that cover the Protein Data Bank (PDB) at the level of 35% sequence identity . With homologous proteins excluded, in 365 cases, the templates identified by our threading program, PROSPECTOR_3, have a root-mean-square deviation (RMSD) to native < 6.5 angstroms, with >70% alignment coverage . After TASSER assembly, in 408 cases the best of the top five full-length models has a RMSD < 6.5 angstroms . Among the 745 targets are 18 membrane proteins, with one-third having a predicted RMSD < 5.5 A . For all representative proteins less than or equal to 300 residues that have corresponding multiple NMR structures in the Protein Data Bank, approximately 20% of the models generated by TASSER are closer to the NMR structure centroid than the farthest individual NMR model . These results suggest that reasonable structure predictions for nonhomologous large size proteins can be automatically generated on a proteomic scale, and the application of this approach to structural as well as functional genomics represent promising applications of TASSER . J Virol, 2004 Oct, 78(20), 10995 - 1006 Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response; Abate DA et al.; We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration . Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response . The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3) . In contrast, NF-kappaB is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus . pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells . pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state . Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response . By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response. Curr Opin Microbiol, 2004 Oct, 7(5), 546 - 54 The promise of functional genomics: completing the encyclopedia of a cell; Hughes TR et al.; Genome sequencing provides complete parts lists of organisms . This presents the obvious challenge of determining how each gene contributes to the life of the organism . This task seems increasingly feasible; however, progress to date suggests that increased interaction between systematic efforts and individual investigators will be critical to completing the encyclopedia of the yeast cell. Curr Opin Microbiol, 2004 Oct, 7(5), 535 - 45 Analyzing protein function on a genomic scale: the importance of gold-standard positives and negatives for network prediction; Jansen R et al.; The concept of 'protein function' is rather 'fuzzy' because it is often based on whimsical terms or contradictory nomenclature . This currently presents a challenge for functional genomics because precise definitions are essential for most computational approaches . Addressing this challenge, the notion of networks between biological entities (including molecular and genetic interaction networks as well as transcriptional regulatory relationships) potentially provides a unifying language suitable for the systematic description of protein function . Predicting the edges in protein networks requires reference sets of examples with known outcome (that is, 'gold standards') . Such reference sets should ideally include positive examples - as is now widely appreciated - but also, equally importantly, negative ones . Moreover, it is necessary to consider the expected relative occurrence of positives and negatives because this affects the misclassification rates of experiments and computational predictions . For instance, a reason why genome-wide, experimental protein-protein interaction networks have high inaccuracies is that the prior probability of finding interactions (positives) rather than non-interacting protein pairs (negatives) in unbiased screens is very small . These problems can be addressed by constructing well-defined sets of non-interacting proteins from subcellular localization data, which allows computing the probability of interactions based on evidence from multiple datasets. Biol Chem, 2004 Aug, 385(8), 691 - 6 Functional genomics identifies novel and diverse molecular targets of nutrients in vivo; Gohil K; Genomic responses to nutrients are important determinants of physiological and pathological functions of living systems . Many of these responses are mediated by changes in mRNA concentrations that are primarily regulated by gene transcription . Transcriptional networks that regulate the expression and activities of transcription factors and structural genes in response to nutrients need to be defined . The tools of functional genomics and bioinformatics offer powerful means to address these needs . The application of global mRNA profiling tools to define genome-wide responses to nutrients and micronutrients with a primary focus on in vivo genomic responses of vital organs of laboratory mice is reviewed here . The studies show that major and minor nutrients affect the expression of mRNAs that are related to aging and inflammation, and chemically diverse micronutrients such as polyphenols and tocopherols may exert their effects through modulating the expression of functionally related genes. Mycol Res, 2004 Aug, 108(Pt 8), 853 - 7 Combining transcriptome data with genomic and cDNA sequence alignments to make confident functional assignments for Aspergillus nidulans genes; Sims AH et al.; Whole genome sequencing of several filamentous ascomycetes is complete or in progress; these species, such as Aspergillus nidulans, are relatives of Saccharomyces cerevisiae . However, their genomes are much larger and their gene structure more complex, with genes often containing multiple introns . Automated annotation programs can quickly identify open reading frames for hypothetical genes, many of which will be conserved across large evolutionary distances, but further information is required to confirm functional assignments . We describe a comparative and functional genomics approach using sequence alignments and gene expression data to predict the function of Aspergillus nidulans genes . By highlighting examples of discrepancies between the automated genome annotation and cDNA or EST sequencing, we demonstrate that the greater complexity of gene structure in filamentous fungi demands independent data on gene expression and the gene sequence be used to make confident functional assignments. Mol Genet Genomics, 2004 Sep, 272(2), 181 - 93 Epub 2004 Jul 30. Mapping Ds insertions in barley using a sequence-based approach; Cooper LD et al.; A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley ( Hordeum vulgaresubsp . vulgare) . The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics . AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation . Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location . The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR . Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population . The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines . In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley . BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences . Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae. J Exp Bot, 2004 Nov, 55(407), 2343 - 51 Epub 2004 Sep 24. Root growth maintenance during water deficits: physiology to functional genomics; Sharp RE et al.; Progress in understanding the network of mechanisms involved in maize primary root growth maintenance under water deficits is reviewed . These include the adjustment of growth zone dimensions, turgor maintenance by osmotic adjustment, and enhanced cell wall loosening . The role of the hormone abscisic acid (ABA) in maintaining root growth under water deficits is also addressed . The research has taken advantage of kinematic analysis, i.e . characterization of spatial and temporal patterns of cell expansion within the root growth zone . This approach revealed different growth responses to water deficits and ABA deficiency in distinct regions of the root tip . In the apical 3 mm region, elongation is maintained at well-watered rates under severe water deficit, although only in ABA-sufficient roots, whereas the region from 3-7 mm from the apex exhibits maximum elongation in well-watered roots, but progressive inhibition of elongation in roots under water deficit . This knowledge has greatly facilitated discovery of the mechanisms involved in regulating the responses . The spatial resolution with which this system has been characterized and the physiological knowledge gained to date provide a unique and powerful underpinning for functional genomics studies . Characterization of water deficit-induced changes in transcript populations and cell wall protein profiles within the growth zone of the maize primary root is in progress . Initial results from EST and unigene analyses in the tips of well-watered and water-stressed roots highlight the strength of the kinematic approach to transcript profiling. Mass Spectrom Rev . 2004 Aug 19; {Epub ahead of print} Mass spectrometry in metabolome analysis; Villas-Boas SG et al.; I.Introduction000 II.Sample Preparation000A . Quenching000B . Extraction000C . Sample Concentration000 III.Analysis000A . GC-MS000 1 . Scope and Resolution of GC-MS000 2 . Analysis of Volatile Metabolites000 3 . Analysis of Semi-Volatile and Non-Volatile Metabolites000 4 . Sensitivity000 5 . Repeatability/Reproducibility of GC-MS for Metabolome Analysis000B . LC-MS000 1 . LC-MS for Target Analysis000 2 . LC-MS for Metabolite Profiling000C . CE-MS000 1 . CE-MS for Target Analysis000 2 . CE-MS for Metabolite Profiling000D . Direct-Infusion Mass Spectrometry000 1 . Direct-Infusion MS for Targeted Analysis000 2 . Direct-Infusion MS for Metabolite Profiling000E . Comparison of the Different Analytical Techniques000 IV.Application on Functional Genomics000 V.Future Outlook000Acknowledgments000References000In the post-genomic era, increasing efforts have been made to describe the relationship between the genome and the phenotype in cells and organisms . It has become clear that even a complete understanding of the state of the genes, messages, and proteins in a living system does not reveal its phenotype . Therefore, researchers have started to study the metabolome (or the metabolic complement of functional genomics) . Within this context, mass spectrometry (MS) has increasingly occupied a central position in the methodologies developed for determination of the metabolic state . This review is mainly focused on the status of MS in the metabolome field, trying to direct the reader to the main approaches for analysis of metabolites, reviewing basic methodologies in sample preparation, and the most recent MS techniques introduced . Apart from the description of the different methods, this review will try to state a general comparison between the several different techniques that involve MS and metabolite analysis, and will highlight their limitations and preferred applicability . Exp Biol Med (Maywood), 2004 Oct, 229(9), 866 - 75 Genomics and clinical medicine: rationale for creating and effectively evaluating animal models; Swanson KS et al.; Because resolving human complex diseases is difficult, appropriate biomedical models must be developed and validated . In the past, researchers have studied diseases either by characterizing a human clinical disease and choosing the most appropriate animal model, or by characterizing a naturally occurring or induced mutant animal and identifying which human disease it best resembled . Although there has been a great deal of progress through the use of these methods, such models have intrinsic faults that limit their relevance to clinical medicine . The recent advent of techniques in molecular biology, genomics, transgenesis, and cloning furnishes investigators with the ability to study vertebrates (e.g., pigs, cows, chickens, dogs) with greater precision and utilize them as model organisms . Comparative and functional genomics and proteomics provide effective approaches for identifying the genetic and environmental factors responsible for complex diseases and in the development of prevention and treatment strategies and therapeutics . By identifying and studying homologous genes across species, researchers are able to accurately translate and apply experimental data from animal experiments to humans . This review supports the hypothesis that associated enabling technologies can be used to create, de novo, appropriate animal models that recapitulate the human clinical manifestation . Comparative and functional genomic and proteomic techniques can then be used to identify gene and protein functions and the interactions responsible for disease phenotypes, which aids in the development of prevention and treatment strategies. Nucleic Acids Res, 2004 Sep 23, 32(17), 4955 - 61 Print 2004. In silico identification of transcriptional regulators associated with c-Myc; Elkon R et al.; The development of powerful experimental strategies for functional genomics and accompanying computational tools has brought major advances in the delineation of transcriptional networks in organisms ranging from yeast to human . Regulation of transcription of eukaryotic genes is to a large extent combinatorial . Here, we used an in silico approach to identify transcription factors (TFs) that form recurring regulatory modules with c-Myc, a protein encoded by an oncogene that is frequently disregulated in human malignancies . A recent study identified, on a genomic scale, human genes whose promoters are bound by c-Myc and its heterodimer partner Max in Burkitt's lymphoma cells . Using computational methods, we identified nine TFs whose binding-site signatures are highly overrepresented in this promoter set of c-Myc targets, pointing to possible functional links between these TFs and c-Myc . Binding sites of most of these TFs are also enriched on the set of mouse homolog promoters, suggesting functional conservation . Among the enriched TFs, there are several regulators known to control cell cycle progression . Another TF in this set, EGR-1, is rapidly activated by numerous stress challenges and plays a central role in angiogenesis . Experimental investigation confirmed that c-Myc and EGR-1 bind together on several target promoters . The approach applied here is general and demonstrates how computational analysis of functional genomics experiments can identify novel modules in complex networks of transcriptional regulation. Rev Med Univ Navarra, 2004 Apr-Jun, 48(2), 36 - 48 {Nutrigenomics and obesity}; Palou A et al.; Obesity is a multifactorial disorder affected by multiple genetic and environmental factors, in particular nutrients, and their interrelationships . Increasing knowledge of the genes and molecules involved in the development of obesity is paving the way for new methods of obesity control . In this sense, Nutrigenomics--which represents a new approach in nutrition research that joints the application of powerful functional genomics technologies, bioinformatics and molecular biology with more traditional methodologies--may orientate the design and development of new functional foods for obesity, based on the scientific knowledge of the impact of specific nutrients on the mammalian body weight control system and their mechanisms of action. Epilepsy Res, 2004 Jul-Aug, 60(2-3), 173 - 8 Molecular profiling of temporal lobe epilepsy: comparison of data from human tissue samples and animal models; Majores M et al.; The advent of gene chip technology and the era of functional genomics have initially been accompanied by huge anticipations to quickly unravel the molecular pathogenesis of multifactorial diseases . Expectations have, today, given way to some concerns about this non-hypothesis driven approach . However, the careful and controlled application of expression microarrays in concert with refined bioinformatic tools may provide novel insights in major disorders particularly of highly complex organs such as the central nervous system (CNS) . Epilepsies are among the most frequent CNS disorders affecting approximately 1.5% of the population worldwide . In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation, a structure located in the mesial temporal lobe . Many TLE patients develop pharmacoresistance, i.e . seizures can no more be controlled by antiepileptic drugs . In order to achieve seizure control, surgical removal of the epileptogenic focus has been established as successful therapeutic strategy . Hippocampal biopsy tissue of pharmacoresistant TLE patients represents an excellent substrate to analyze molecular mechanisms related to structural and cellular reorganization in epilepsy . The complexity of alterations in TLE hippocampi suggests numerous genes and signaling cascades to be involved in the pathogenesis . By microarrays, genome wide expression profiles can be constituted from TLE tissues . However, hippocampi of pharmacoresistant TLE patients represent an advanced stage of the disease . Early stages of epilepsy development are not available for functional genome analysis in humans . Animal models of TLE appear particularly helpful to study molecular mechanisms of highly dynamic processes such as the development of hyperexcitability and pharmacoresistance . In this review, we summarize recent data of gene expression profiles in human and experimental TLE and discuss the relevance of novel tools for bioinformatic analysis and data mining. Curr Opin Genet Dev, 2004 Oct, 14(5), 470 - 6 Genome-wide high-throughput screens in functional genomics; Friedman A et al.; The availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function . Within the past year, rapid advances have been made towards this goal in many major model systems, including yeast, worms, flies, and mammals . Yeast genome-wide screens have taken advantage of libraries of deletion strains, but RNA-interference has been used in other organisms to knockdown gene function . Examples of recent large-scale functional genetic screens include drug-target identification in yeast, regulators of fat accumulation in worms, growth and viability in flies, and proteasome-mediated degradation in mammalian cells . Within the next five years, such screens are likely to lead to annotation of function of most genes across multiple organisms . Integration of such data with other genomic approaches will extend our understanding of cellular networks. Mol Genet Genomics, 2004 Oct, 272(3), 344 - 52 Epub 2004 Sep 16. Efficient gene disruption in the koji-mold Aspergillus sojae using a novel variation of the positive-negative method; Takahashi T et al.; When no phenotypic screen is available, gene disruption in the koji-mold Aspergillus sojae is a time-consuming process, owing to the low frequency of homologous recombination . To achieve efficient gene disruption in the koji-mold, we developed a novel positive-negative selection method to enrich for homologous recombinants . The pyrG gene from A . sojae was used as a positive selection marker for transformants, and the oliC31 gene of A . nidulans, which codes for a mutant form of subunit 9 of the F1FO-ATPase, was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants . The positive-negative selection markers, in combination with a pyrG deletion strain as a host, enabled enrichment for homologous recombinants, and disruption of the genes niaD, areA and tannase was successfully demonstrated . In order to examine whether the positive-negative selection technique is effective for targeting any locus, even in the absence of information on gene function or phenotype, we attempted to disrupt the aflR gene of A . sojae, which codes for a putative transcription factor for the aflatoxin biosynthetic pathway, using the method . Despite the fact that this gene is not transcribed in A . sojae, aflR disruptants were efficiently obtained, suggesting that the method is indeed capable of targeting any locus, without additional ectopic integration, and is thus applicable for functional genomics studies in filamentous fungi, including A . sojae. Mol Genet Genomics, 2004 Oct, 272(3), 308 - 27 Epub 2004 Sep 11. Genetic mapping of EST-derived microsatellites from the diploid Gossypium arboreum in allotetraploid cotton; Han ZG et al.; To increase the numbers of microsatellites available for use in constructing a genetic map, and facilitate the use of functional genomics to elucidate fiber development and breeding in cotton, we sampled microsatellite sequences from expressed sequence tags (ESTs) transcribed during fiber elongation in the A-genome species Gossypium arboreum to evaluate their frequency of occurrence, level of polymorphism and distribution in the At and Dt subgenomes of tetraploid cotton . From among ESTs derived from G . arboreum fibers at 7-10 days post anthesis (dpa), 931 ESTs were found to contain simple sequence repeats (SSRs); 544 (58.4%) EST-SSR primer pairs were developed, and 468 (86%) amplified PCR products from allotetraploid cotton (G . hirsutumcv . TM-1 and G . barbadense cv . Hai7124) . However, only 99 (18.2%) of these were found to be polymorphic and segregating in our interspecific BC1 mapping population {(TM-1xHai7124)xTM-1} . In these amplified and informative EST-SSRs, hexa- and tri-nucleotide repeat motifs were the most frequent, representing 40.1 and 30%, respectively, of the total . A total of 111 loci detected with these 99 EST-SSRs were integrated into our backbone map including 511 SSR loci . The distribution of the EST-SSRs appeared to be non-random, since 72 loci were anchored to the At and 37 to the Dt subgenome of allotetraploid cotton based on linkage tests . Interestingly, out of the 10 pairs of duplicate loci amplified, seven were mapped to the corresponding homologous linkage groups and/or chromosomes . BLASTX analysis revealed that 69 of the 99 ESTs showed significant similarities to known genes . Some genes important for fiber development, such as sucrose synthase, were mapped to corresponding chromosomes . These EST-SSRs provide structural and functional genomic information that will be useful for understanding cotton fiber development. Med Sci (Paris), 2004 Aug-Sep, 20(8-9), 815 - 9 {RNA interference: towards a functional genomics in mammalian cells?}; Dautry F et al.; The discovery of the induction of RNA degradation by double stranded RNA in C . elegans, "RNA interference", makes it possible to envision systematic studies of gene function in mammalian cells . Indeed, in spite of the existence in mammals of the interferon response to double stranded RNA, the introduction of small interfering RNA can induce a sequence specific inhibition of gene expression either through RNA degradation or by blocking translation . Although the inhibition is transient and usually not complete, strategies have been developed to achieve long term gene silencing . The issue of target specificity is still not completely clear and will probably constitute a limitation of this approach . However, because of the unprecedented ease with which large scale screens can be performed, RNA interference has already established itself as the tool of choice to initiate functional genomics in mammalian cells. Bioinformatics . 2004 Sep 9; {Epub ahead of print} VizRank: finding informative data projections in functional genomics by machine learning; Leban G et al.; SUMMARY: VizRank is a tool that finds interesting two-dimensional projections of class-labeled data . When applied to multi-dimensional functional genomics data sets, VizRank can systematically find relevant biological patterns . AVAILABILITY: SUPPLEMENTARY INFORMATION: http://www.ailab.si/supp/bi-vizrank. Trends Endocrinol Metab, 2004 Aug, 15(6), 243 - 7 Flying through the genome: a comprehensive study of functional genomics using RNAi in Drosophila; Andres AJ; Sequencing the DNA of an entire mammalian genome now seems routine . The human sequence along with the mouse--the model for mammalian genetics--and the rat--the model for mammalian physiology--are now part of the data archive . However, the real challenges for the 21st century are what to do with this information and how to test the function of so many different genes in so many different cellular contexts . The potential payoffs are enormous . Examples include a better understanding of disease pathologies with effective strategies for therapeutic interventions that cause few, if any, side effects. Trends Plant Sci, 2004 Aug, 9(8), 391 - 8 Weed genomics: new tools to understand weed biology; Basu C et al.; In spite of the large yield losses that weeds inflict on crops, we know little about the genomics of economically important weed species . Comparative genomics between plant model species and weeds, map-based approaches, genomic sequencing and functional genomics can play vital roles in understanding and dissecting weedy traits of agronomically important weed species that damage crops . Weed genomics research should increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make weeds a successful group of plants . Here, we propose specific weed candidates as genomic models, including economically important plants that have evolved herbicide resistance on several occasions and weeds with good comparative genomic qualities that can be anchored to the genomics of Arabidopsis and Oryza sativa. BMC Genomics . 2004 Sep 09;5(1):64. A method for detecting and correcting feature misidentification on expression microarrays; Tu IP et al.; BACKGROUND: Much of the microarray data published at Stanford is based on mouse and human arrays produced under controlled and monitored conditions at the Brown and Botstein laboratories and at the Stanford Functional Genomics Facility (SFGF) . Nevertheless, as large datasets based on the Stanford Human array began to accumulate, a small but significant number of discrepancies were detected that required a serious attempt to track down the original source of error . Due to a controlled process environment, sufficient data was available to accurately track the entire process leading to up to the final expression data . In this paper, we describe our statistical methods to detect the inconsistencies in microarray data that arise from process errors, and discuss our technique to locate and fix these errors . RESULTS: To date, the Brown and Botstein laboratories and the Stanford Functional Genomics Facility have together produced 40,000 large-scale (10-50,000 feature) cDNA microarrays . By applying the heuristic described here, we have been able to check most of these arrays for misidentified features, and have been able to confidently apply fixes to the data where needed . Out of the 265 million features checked in our database, problems were detected and corrected on 1.3 million of them . CONCLUSION: Process errors in any genome scale high throughput production regime can lead to subsequent errors in data analysis . We show the value of tracking multi-step high throughput operations by using this knowledge to detect and correct misidentified data on gene expression microarrays. Pigment Cell Res, 2004 Oct, 17(5), 461 - 70 Functional genomics tools for the analysis of zebrafish pigment; Pickart MA et al.; Genetic model organisms are increasingly valuable in the post-genomics era to provide a basis for comparative analysis of the human genome . For higher order processes of vertebrate pigment cell biology and development, the mouse has historically been the model of choice . A complementary organism, the zebrafish (Danio rerio), shares many of the signaling and biological processes of vertebrates, e.g . neural crest development . The zebrafish has a number of characteristics that make it an especially valuable model for the study of pigment cell biology and disease . Large-scale genetic screens have identified a collection of pigmentation mutants that have already made valuable contributions to pigment research . An increasing repertoire of genomic resources such as an expressed sequence tag-based Gene Index (The Institute for Genomic Research) and improving methods of mutagenesis, transgenesis, and gene targeting make zebrafish a particularly attractive model . Morpholino phosphorodiamidate oligonucleotide (MO) 'knockdown' of pigment gene expression provides a non-conventional antisense tool for the analysis of genes involved in pigment cell biology and disease . In addition, an ongoing, reverse-genetic, MO-based screen for the rapid identification of gene function promises to be a valuable complement to other high-throughput microarray and proteomic approaches for understanding pigment cell biology . Novel reagents for zebrafish transgenesis, such as the Sleeping Beauty transposon system, continue to improve the capacity for genetic analysis in this system and ensure that the zebrafish will be a valuable genetic model for understanding a variety of biological processes and human diseases for years to come. Proc Natl Acad Sci U S A, 2004 Sep 21, 101(38), 13951 - 6 Epub 2004 Sep 07. A Populus EST resource for plant functional genomics; Sterky F et al.; Trees present a life form of paramount importance for terrestrial ecosystems and human societies because of their ecological structure and physiological function and provision of energy and industrial materials . The genus Populus is the internationally accepted model for molecular tree biology . We have analyzed 102,019 Populus ESTs that clustered into 11,885 clusters and 12,759 singletons . We also provide >4,000 assembled full clone sequences to serve as a basis for the upcoming annotation of the Populus genome sequence . A public web-based EST database (POPULUSDB) provides digital expression profiles for 18 tissues that comprise the majority of differentiated organs . The coding content of Populus and Arabidopsis genomes shows very high similarity, indicating that differences between these annual and perennial angiosperm life forms result primarily from differences in gene regulation . The high similarity between Populus and Arabidopsis will allow studies of Populus to directly benefit from the detailed functional genomic information generated for Arabidopsis, enabling detailed insights into tree development and adaptation . These data will also valuable for functional genomic efforts in Arabidopsis. Proteomics, 2004 Sep, 4(9), 2672 - 84 Wheat leaf proteome analysis using sequence data of proteins separated by two-dimensional electrophoresis; Bahrman N et al.; Identifying wheat leaf protein expression is a major challenge of functional genomics . Using two-dimensional gel electrophoresis 541 wheat leaf proteins were separated and 55 of them were sequenced by nano liquid chromatography-tandem mass spectrometry . Peptide sequence data were screened against protein banks and expressed sequence tag public banks . Among these 55 spots, 20 proteins were found in wheat and 21 in other grass families . Twelve proteins showed similarities with other eukaryotic plant species . One protein showed homology to a bacterial sequence and another protein remained unknown . In 18 cases a significant score was found for the wheat TUC (Tentative Unique Contigs) of the PlantGDB data . In several cases, different spots were identified as corresponding to the same protein that can probably be attributed to the hexaploid structure of wheat . The identified proteins were classified in six groups and