Despite continual progress in the cataloging of vertebrate regulatory elements, little is known about their organization and regulatory architecture. Here we describe a massively parallel experiment to systematically test the impact of copy number, spacing, combination and order of transcription factor binding sites on gene expression. A complex library of ~5,000 synthetic regulatory elements containing patterns from 1 2 liver-specific transcription factor binding sites was assayed in mice and in HepG2 cells. We find that certain transcription factors act as direct drivers of gene expression in homotypic clusters of binding sites, independent of spacing between sites, whereas others function only synergistically. Heterotypic enhancers are stronger than their homotypic analogs and favor specific transcription factor binding site combinations, mimicking putative native enhancers. Exhaustive testing of binding site permutations suggests that there is flexibility in binding site order. Our findings provide quantitative support for a flexible model of regulatory element activity and suggest a framework for the design of synthetic tissue-specific enhancers.
Regulatory elements play an important role in the variability of individual responses to drug treatment. This has been established through studies on three classes of elements that regulate RNA and protein abundance: promoters, enhancers and microRNAs. Each of these elements, and genetic variants within them, are being characterized at an exponential pace by next-generation sequencing (NGS) technologies. In this review, we outline examples of how each class of element affects drug response via regulation of drug targets, transporters and enzymes. We also discuss the impact of NGS technologies such as chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq), and the ramifications of new techniques such as high-throughput chromosome capture (Hi-C), chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and massively parallel reporter assays (MPRA). NGS approaches are generating data faster than they can be analyzed, and new methods will be required to prioritize laboratory results before they are ready for the clinic. However, there is no doubt that these approaches will bring about a systems-level understanding of the interplay between genetic variants and drug response. An understanding of the importance of regulatory variants in pharmacogenomics will facilitate the identification of responders versus non-responders, the prevention of adverse effects and the optimization of therapies for individual patients.
ChIP-Seq; enhancers; miRNA; next-generation sequencing; pharmacogenomics; promoters; RNA-Seq
We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.
How should funding agencies enable researchers to explore high-risk but potentially high-reward science? One model that appears to work is the NSF-funded synthesis center, an incubator for community-led, innovative science.
Mutualistic symbioses between scleractinian corals and endosymbiotic dinoflagellates (Symbiodinium spp.) are the foundation of coral reef ecosystems. For many coral-algal symbioses, prolonged episodes of thermal stress damage the symbiont's photosynthetic capability, resulting in its expulsion from the host. Despite the link between photosynthetic competency and symbiont expulsion, little is known about the effect of thermal stress on the expression of photosystem genes in Symbiodinium. This study used real-time PCR to monitor the transcript abundance of two important photosynthetic reaction center genes, psbA (encoding the D1 protein of photosystem II) and psaA (encoding the P700 protein of photosystem I), in four cultured isolates (representing ITS2-types A13, A20, B1, and F2) and two in hospite Symbiodinium spp. within the coral Pocillopora spp. (ITS2-types C1b-c and D1). Both cultured and in hospite Symbiodinium samples were exposed to elevated temperatures (32°C) over a 7-day period and examined for changes in photochemistry and transcript abundance. Symbiodinium A13 and C1b-c (both thermally sensitive) demonstrated significant declines in both psbA and psaA during the thermal stress treatment, whereas the transcript levels of the other Symbiodinium types remained stable. The downregulation of both core photosystem genes could be the result of several different physiological mechanisms, but may ultimately limit repair rates of photosynthetic proteins, rendering some Symbiodinium spp. especially susceptible to thermal stress.
Mesenchymal stem cells (MSCs) have the potential to replace or restore the function of damaged tissues and offer much promise in the successful application of tissue engineering and regenerative medicine strategies. Optimising culture conditions for the pre-differentiation of MSCs is a key goal for the research community, and this has included a number of different approaches, one of which is the use of mechanical stimuli. Mesenchymal tissues are subjected to mechanical stimuli in vivo and terminally differentiated cells from the mesenchymal lineage respond to mechanical stimulation in vivo and in vitro. MSCs have also been shown to be highly mechanosensitive and this may present an ideal method for controlling MSC differentiation. Here we present an overview of the response of MSCs to various mechanical stimuli, focusing on their differentiation towards the mesenchymal tissue lineages including bone, cartilage, tendon/ligament, muscle and adipose tissue. More research is needed to elucidate the complex interactions between biochemically and mechanically stimulated differentiation pathways.
mechanical stimuli; mesenchymal stem cell; osteogenesis; tenogenesis
The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian liver enhancers at single nucleotide resolution via a massively parallelized reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2–3% divergence from wild-type. Each haplotype was linked to a unique sequence tag embedded within a transcriptional cassette. We introduced each enhancer library into mouse liver and measured the relative activities of individual haplotypes en masse by sequencing of the transcribed tags. Linear regression yielded highly reproducible estimates of the impact of every possible single nucleotide change on enhancer activity. The functional impact of most mutations was modest, with ~22% impacting activity by >1.2-fold, and only ~3% by >2-fold. These results suggest that mammalian enhancers are relatively robust to single nucleotide changes. Several, but not all positions with higher impact showed evidence for purifying selection, or co-localized with known liver-associated transcription factor binding sites, demonstrating the value of empirical high-resolution functional analysis.
Many low- and middle-income countries (LMICs) lack basic surgical resources, resulting in avoidable disability and mortality. Recently, residents in surgical training programs have shown increasing interest in overseas elective experiences to assist surgical programs in LMICs. The purpose of this study was to survey Canadian surgical residents about their interest in international volunteerism.
We sent a web-based survey to all general and orthopedic surgery residents enrolled in surgical training programs in Canada. The survey assessed residents’ interests, attitudes and motivations, and perceived barriers and aids with respect to international volunteerism.
In all, 361 residents completed the survey for a response rate of 38.0%. Half of the respondents indicated that the availability of an international surgery elective would have positively influenced their selection of a residency program. Excluding the 18 residents who had volunteered during residency, 63.8% of the remaining residents confirmed an interest in international volunteering with “contributing to an important cause,” “teaching” and “tourism/cultural enhancement” as the leading reasons for their interest. Perceived barriers included “lack of financial support” and “lack of available organized opportunities.” All (100%) respondents who had done an international elective during residency confirmed that they would pursue such work in the future.
Administrators of Canadian surgical programs should be aware of strong resident interest in global health care and accordingly develop opportunities by encouraging faculty mentorships and resources for global health teaching.
Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS’s enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.
High-throughput screening data repositories, such as PubChem, represent valuable resources for the development of small molecule chemical probes and can serve as entry points for drug discovery programs. While the loose data format offered by PubChem allows for great flexibility, important annotations, such as the assay format and technologies employed, are not explicitly indexed. We have previously developed a BioAssay Ontology (BAO) and curated over 350 assays with standardized BAO terms. Here we describe the use of BAO annotations to analyze a large set of assays that employ luciferase- and β-lactamase-based technologies. We identified promiscuous chemotypes pertaining to different sub-categories of assays and specific mechanisms by which these chemotypes interfere in reporter gene assays. Our results show that the data in PubChem can be used to identify promiscuous compounds that interfere non-specifically with particular technologies. Furthermore, we show that BAO is a valuable toolset for the identification of related assays and for the systematic generation of insights that are beyond the scope of individual assays or screening campaigns.
compound promiscuity; assay ontology; reporter gene assays; high-throughput screening data analysis; cheminformatics
MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.
The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.
More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.
Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.
Breast cancer; miRNA; Biomarker; Neoadjuvant chemotherapy; Metastasis
Neurons in the peripheral nervous system (PNS) display a higher capacity to regenerate after injury than those in the central nervous system, suggesting cell specific transcriptional modules underlying axon growth and inhibition. We report a systems biology based search for PNS specific transcription factors (TFs). Messenger RNAs enriched in dorsal root ganglion (DRG) neurons compared to cerebellar granule neurons (CGNs) were identified using subtractive hybridization and DNA microarray approaches. Network and transcription factor binding site enrichment analyses were used to further identify TFs that may be differentially active. Combining these techniques, we identified 32 TFs likely to be enriched and/or active in the PNS. Twenty-five of these TFs were then tested for an ability to promote CNS neurite outgrowth in an overexpression screen. Real-time PCR and immunohistochemical studies confirmed that one representative TF, STAT3, is intrinsic to PNS neurons, and that constitutively active STAT3 is sufficient to promote CGN neurite outgrowth.
Dorsal Root Ganglion; Transcription Factor; High Content Analysis; Screen; Systems Biology; Cerebellar Granule Neuron; STAT3
Mutualisms between reef-building corals and endosymbiotic dinoflagellates are particularly sensitive to environmental stress, yet the ecosystems they construct have endured major oscillations in global climate. During the winter of 2008, an extreme cold-water event occurred in the Gulf of California that bleached corals in the genus Pocillopora harbouring a thermally ‘sensitive’ symbiont, designated Symbiodinium C1b-c, while colonies possessing Symbiodinium D1 were mostly unaffected. Certain bleached colonies recovered quickly while others suffered partial or complete mortality. In most colonies, no appreciable change was observed in the identity of the original symbiont, indicating that these partnerships are stable. During the initial phases of recovery, a third species of symbiont B1Aiptasia, genetically identical to that harboured by the invasive anemone, Aiptasia sp., grew opportunistically and was visible as light-yellow patches on the branch tips of several colonies. However, this symbiont did not persist and was displaced in all cases by C1b-c several months later. Colonies with D1 were abundant at inshore habitats along the continental eastern Pacific, where seasonal turbidity is high relative to offshore islands. Environmental conditions of the central and southern coasts of Mexico were not sufficient to explain the exclusivity of D1 Pocillopora in these regions. It is possible that mass mortalities associated with major thermal disturbances during the 1997–1998 El Niño Southern Oscillation eliminated C1b-c holobionts from these locations. The differential loss of Pocillopora holobionts in response to thermal stress suggests that natural selection on existing variation can cause rapid and significant shifts in the frequency of particular coral–algal partnerships. However, coral populations may take decades to recover following episodes of severe selection, thereby raising considerable uncertainty about the long-term viability of these communities.
climate change; coral bleaching; eastern Pacific; holobiont; natural selection; Symbiodinium
Fibromyalgia is a troubling disease characterized by chronic pain. This study explored whether pain and other fibromyalgia symptoms are worse among women who had undergone a hysterectomy with or without an oophorectomy versus those who had not.
Consecutive women who were seen at the Fibromyalgia Treatment Program at a tertiary medical center between 2001 and 2004 and who completed the Fibromyalgia Impact Questionnaire (FIQ) and Short Form-36 Health Survey (SF-36) at initial evaluation were included in this study.
A total of 813 women were included; 328 had had a hysterectomy. Total FIQ scores from women who had had a hysterectomy were higher (worse symptoms) than those who had not (58.1 vs 56.4, P = 0.002). FIQ subscale scores of pain (P = 0.003), fatigue (P = 0.030), stiffness (P = 0.035), and depression (P = 0.008) were also worse in women who had had a hysterectomy. Similar to the FIQ, SF-36 physical component scores were worse in women who had had a hysterectomy (P = 0.045).
Pain and other fibromyalgia symptom severity was worse in women who had had a hysterectomy with or without an oophorectomy.
fibromyalgia; hysterectomy; oophorectomy; symptom severity; surgical menopause
High-throughput screening (HTS) is one of the main strategies to identify novel entry points for the development of small molecule chemical probes and drugs and is now commonly accessible to public sector research. Large amounts of data generated in HTS campaigns are submitted to public repositories such as PubChem, which is growing at an exponential rate. The diversity and quantity of available HTS assays and screening results pose enormous challenges to organizing, standardizing, integrating, and analyzing the datasets and thus to maximize the scientific and ultimately the public health impact of the huge investments made to implement public sector HTS capabilities. Novel approaches to organize, standardize and access HTS data are required to address these challenges.
We developed the first ontology to describe HTS experiments and screening results using expressive description logic. The BioAssay Ontology (BAO) serves as a foundation for the standardization of HTS assays and data and as a semantic knowledge model. In this paper we show important examples of formalizing HTS domain knowledge and we point out the advantages of this approach. The ontology is available online at the NCBO bioportal http://bioportal.bioontology.org/ontologies/44531.
After a large manual curation effort, we loaded BAO-mapped data triples into a RDF database store and used a reasoner in several case studies to demonstrate the benefits of formalized domain knowledge representation in BAO. The examples illustrate semantic querying capabilities where BAO enables the retrieval of inferred search results that are relevant to a given query, but are not explicitly defined. BAO thus opens new functionality for annotating, querying, and analyzing HTS datasets and the potential for discovering new knowledge by means of inference.
Neurons in the central nervous system lose their intrinsic capacity for axon regeneration as they mature, and it is widely hypothesized that changes in gene expression are responsible. Testing this hypothesis and identifying the relevant genes has been challenging because hundreds to thousands of genes are developmentally regulated in CNS neurons, but only a small subset are likely relevant to axon growth. Here we used automated high content analysis (HCA) methods to functionally test 743 plasmids encoding developmentally regulated genes in neurite outgrowth assays using postnatal cortical neurons. We identified both growth inhibitors (Ephexin, Aldolase A, Solute Carrier 2A3, and Chimerin), and growth enhancers (Doublecortin, Doublecortin-like, Kruppel-like Factor 6, and CaM-Kinase II gamma), some of which regulate established growth mechanisms like microtubule dynamics and small GTPase signaling. Interestingly, with only one exception the growth-suppressing genes were developmentally upregulated, and the growth-enhancing genes downregulated. These data provide important support for the hypothesis that developmental changes in gene expression control neurite outgrowth, and identify potential new gene targets to promote neurite outgrowth.
Humans with L1 cell adhesion molecule (L1CAM) mutations exhibit X-linked hydrocephalus, as well as other severe neurological disorders. L1-6D mutant mice, which are homozygous for a deletion that removes the sixth immunoglobulin-like domain of L1cam, seldom display hydrocephalus on the 129/Sv background. However, the same L1-6D mutation produces severe hydrocephalus on the C57BL/6J background. To begin to understand how L1cam deficiencies result in hydrocephalus and to identify modifier loci that contribute to X-linked hydrocephalus by genetically interacting with L1cam, we conducted a genome-wide scan on F2 L1-6D mice, bred from L1-6D 129S2/SvPasCrlf and C57BL/6J mice. Linkage studies, utilizing chi-square tests and quantitative trait loci mapping techniques, were performed. Candidate modifier loci were further investigated in an extension study. Linkage was confirmed for a locus on chromosome 5, which we named L1cam hydrocephalus modifier 1 (L1hydro1), p = 4.04 × 10−11.
L1cam; Hydrocephalus; Modifier; Linkage analysis; QTL
Reef corals are sentinels for the adverse effects of rapid global warming on the planet's ecosystems. Warming sea surface temperatures have led to frequent episodes of bleaching and mortality among corals that depend on endosymbiotic micro-algae (Symbiodinium) for their survival. However, our understanding of the ecological and evolutionary response of corals to episodes of thermal stress remains inadequate. For the first time, we describe how the symbioses of major reef-building species in the Caribbean respond to severe thermal stress before, during and after a severe bleaching event. Evidence suggests that background populations of Symbiodinium trenchi (D1a) increased in prevalence and abundance, especially among corals that exhibited high sensitivity to stress. Contrary to previous hypotheses, which posit that a change in symbiont occurs subsequent to bleaching, S. trenchi increased in the weeks leading up to and during the bleaching episode and disproportionately dominated colonies that did not bleach. During the bleaching event, approximately 20 per cent of colonies surveyed harboured this symbiont at high densities (calculated at less than 1.0% only months before bleaching began). However, competitive displacement by homologous symbionts significantly reduced S. trenchi's prevalence and dominance among colonies after a 2-year period following the bleaching event. While the extended duration of thermal stress in 2005 provided an ecological opportunity for a rare host-generalist symbiont, it remains unclear to what extent the rise and fall of S. trenchi was of ecological benefit or whether its increased prevalence was an indicator of weakening coral health.
Caribbean; climate change; competitive displacement; coral bleaching; opportunistic species; Symbiodinium
The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and α-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome.
difference in gel electrophoresis (DIGE); mitochondria; nitric oxide (NO); redox signalling; S-nitrosation; S-nitrosylation; 2D, two-dimensional; ALDH2, aldehyde dehydrogenase; AR, area at risk; BVA, biological variance analysis; Cy3, indocarbocyanine; Cy5, indodicarbocyanine; DIGE, difference in gel electrophoresis; DTT, dithiothreitol; GSNO, S-nitrosoglutathione; I/R, ischaemia/reperfusion; α-KGDH, α-ketoglutarate dehydrogenase; LAD, left anterior descending coronary artery; LV, left ventricle; MALDI–TOF-TOF, matrix-assisted laser-desorption ionization–time-of-flight time-of-flight; MitoNAP, mito-N-acetylpenicillamine; MitoSNO, mitochondria-targeted S-nitrosothiol; NEM, N-ethylmaleimide; NIH, National Institutes for Health; PrSNO, protein S-nitrosothiol; RHM, rat heart mitochondria; RLM, rat liver mitochondria; ROS, reactive oxygen species; SA, standardized abundance; SNAP, S-nitroso-N-acetylpenicillamine; SNO-DIGE, S-nitrosothiol difference in gel electrophoresis; TPP, triphenylphosphonium
Cancer cells are frequently glycolytic and over-express hexokinase II (HXK II). In cancer cells, the majority of hexokinase II is localized to the mitochondria through interaction with the voltage dependent anion channel (VDAC). Disruption in the binding of hexokinase II to the mitochondria has been shown to promote mitochondrial injury provoked by pro-apoptotic proteins.
The present study demonstrates that cisplatin induces the PIDD (P53 induced protein with a death domain) dependent activation of caspase-2. In turn, caspase-2 cleaves and activates Bid, resulting in the oligomerization of Bak and the release of cytochrome c. Notably, the detachment of hexokinase II from the mitochondria markedly potentiates the onset of caspase-2 induced mitochondrial damage, thus resulting in a synergistic induction of cisplatin induced cytotoxicity.
Hexokinase II; caspase-2; cisplatin; mitochondria
Piperidine nitroxides such as TEMPOL have been widely used as antioxidants in vitro and in vivo. MitoTEMPOL is a mitochondria-targeted derivative of TEMPOL designed to protect mitochondria from the oxidative damage that they accumulate, but once there is rapidly reduced to its hydroxylamine, MitoTEMPOL-H. As little is known about the antioxidant efficacy of hydroxylamines, this study has assessed the antioxidant activity of both MitoTEMPOL and MitoTEMPOL-H. The hydroxylamine was more effective at preventing lipid-peroxidation than MitoTEMPOL and decreased oxidative damage to mitochondrial DNA caused by menadione. In contrast to MitoTEMPOL, MitoTEMPOL-H has no superoxide dismutase activity and its antioxidant actions are likely to be mediated by hydrogen atom donation. Therefore, even though MitoTEMPOL is rapidly reduced to MitoTEMPOL-H in cells, it remains an effective antioxidant. Furthermore, as TEMPOL is also reduced to a hydroxylamine in vivo, many of its antioxidant effects may also be mediated by its hydroxylamine.
MitoTEMPOL; nitroxide; hydroxylamine; lipid peroxidation; mtDNA; antioxidant
Robin Smith, MD, MBA, isn't claiming she has found the fountain of youth, but she is certain her company, NeoStem, can help prevent many costly, debilitating scourges of older age.
Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds.
We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples) produced by subtractive hybridization.
EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects.
Three living male darwinulid ostracods of a new species of the genus Vestalenula have been found in Yakushima, Japan. This is the first report of living darwinulid males for over 100 years and their morphology casts doubt on the two previous records from the late 1800s. The presence of male darwinulids also calls into question the hypothesis that the family Darwinulidae is an exclusively ancient asexual group, reproducing without sex for over 200 million years (Myr). Male carapaces are of similar size and shape to A-1 juvenile females of the same species, suggesting that males may have been dismissed as A-1 juveniles in other living and fossil species. The antennae and fifth limbs are sexually dimorphic: the male antennae have six segments compared with five in the female and a series of putative chemical receptors originating at the extra segment boundary, while the male fifth limbs have well-developed grasping hooks, as in males of many ostracod groups. The lack of Zenker's Organ and of complex internal structures within the hemipenis contradicts previous hypotheses of the phylogenetic position of darwinulids.
Darwinulidae; males; sexual dimorphism; Ostracoda; Vestalenula
Members of the Collaborative Immunization Initiatives determined the immunization coverage rates for two groups of children in our clinic: those 7 to 12 months old and those 18 to 23 months old. The Clinic Assessment Software Application from the Centers for Disease Control and Prevention was used. The immunization rates determined by this method appeared to significantly underestimate the vaccination coverage rates in our clinic. A review of available charts included in the original sample was done excluding patients no longer attending our clinic. We found a higher rate of coverage in the same sample and a low rate of missed opportunities for administering immunizations. The major reason for this discrepancy is overly stringent Clinic Assessment Software Application inclusion criteria. Additional factors include failure to take into account the wide range of acceptable ages for administering immunizations and different dosages for different brands of vaccines. Different methods of calculation may cause as much as a 20% difference in immunization rates for the same or similar population groups. Such large differences may lead to vastly different responses and interventions. We believe that a central registry is the most accurate method of determining immunization rates. Until this is widely available and applied, a more accurate measure of a facility’s immunization effectiveness is the number of missed opportunities for administering immunizations.
Immunization; Immunization Programs; Immunization Rates; Patient Participation Rates; Registries; Vaccination