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PubMed2003

An investigation of error sources and their impact in estimating the time to the most recent ancestor of spatially and temporally distributed HIV sequences.

This is an investigation of significant error sources and their impact in estimating the time to the most recent common ancestor (MRCA) of spatially and temporally distributed human immunodeficiency virus (HIV) sequences. We simulate an HIV epidemic under a range of assumptions with known time to the MRCA (tMRCA). We then apply a range of baseline (known) evolutionary models to generate sequence data. We next estimate or assume one of several misspecified models and use the chosen model to estimate the time to the MRCA. Random effects and the extent of model misspecification determine the magnitude of error sources that could include: neglected heterogeneity in substitution rates across lineages and DNA sites; uncertainty in HIV isolation times; uncertain magnitude and type of population subdivision; uncertain impacts of host/viral transmission dynamics, and unavoidable model estimation errors. Our results suggest that confidence intervals will rarely have the nominal coverage probability for tMRCA. Neglected effects lead to errors that are unaccounted for in most analyses, resulting in optimistically narrow confidence intervals (CI). Using real HIV sequences having approximately known isolation times and locations, we present possible confidence intervals for several sets of assumptions. In general, we cannot be certain how much to broaden a stated confidence interval for tMRCA. However, we describe the impact of candidate error sources on CI width. We also determine which error sources have the most impact on CI width and demonstrate that the standard bootstrap method will underestimate the CI width.

Animals↗

PubMed1997

Stem cell gene therapy, position effects and chromatin insulators.

Low efficiency of gene transfer is the main obstacle for a clinically effective gene therapy at the level of the pluripotent hematopoietic stem cell. Another important aspect of stem cell gene therapy, the actual expression of the transduced genes, has only been investigated adequately in very few studies, mainly for globin genes. Transcriptional silencing and position effects due to negative effects of surrounding chromatin on the expression of randomly integrated vector sequences may seriously jeopardize the success of current gene therapy strategies, even if transduction efficiency can be significantly improved. We propose the incorporation of chromatin insulators in the design of gene therapy vectors to overcome the problem of position effects. Chromatin insulators are protein-binding DNA elements that lack intrinsic promoter/enhancer activity but shelter genes from transcriptional influence of surrounding chromatin. The best characterized insulators are from Drosophila. We hypothesize that the important cellular function of chromatin organization is evolutionarily conserved and that human homologs to Drosophila insulator binding proteins such as the suppressor of Hairy-wing exist and can be cloned. Using these putative proteins, it should be possible to identify corresponding minimal binding sites with insulator activity. The design and incorporation of effective chromatin insulator sequences in the next generation of gene therapy vectors should lead to improved and more predictable expression of therapeutic transgenes and constitute an important step toward clinically effective gene therapy.

Animals↗

PubMed2006

Annotation, nomenclature and evolution of four novel homeobox genes expressed in the human germ line.

The homeobox genes comprise a large gene superfamily characterised by a conserved DNA motif encoding the homeodomain. Most homeodomain proteins function as transcription factors, and many have important roles in embryonic development and cell differentiation. Here we describe, annotate and name four novel homeobox genes in the human genome: ARGFX, DPRX, TPRX1 and DUXA. Each has generated multiple retrotransposed (processed) pseudogenes; these are reliable indicators of germ-line expression because only in germ-line cells can retrotransposition result in inheritance to the next generation. The retrotransposed sequences were exploited here as a novel means to deduce exon-intron boundaries. All four novel genes show accelerated rates of protein sequence evolution. This fast rate of sequence change may be connected with roles in human reproductive biology. Deducing the evolutionary origins of these genes is not straightforward, but we propose that TPRX1, DPRX and DUXA are highly divergent derivatives of the CRX gene, itself a member of the Otx homeobox gene family.

Evolution, Molecular↗

PubMed2026

Association between smoking, tumor genetics, and outcomes in men with metastatic prostate cancer.

PURPOSE: Smoking has been associated with increased metastatic prostate cancer mortality, but the mechanisms behind this are largely unknown. We hypothesized that smoking increases the risk of genetic alterations associated with aggressive disease and/or the transformation to neuroendocrine prostate cancer (NEPC). PATIENTS AND METHODS: We utilized the Prostate Cancer Precision Medicine Multi-institutional Collaborative Effort (PROMISE) clinical genomic database for this retrospective analysis. We associated patient characteristics and tumor genetic data with smoking exposure at diagnosis (current, former, never and pack years) and with clinical outcomes, including overall survival (OS) from diagnosis or time to developing metastatic disease and NEPC status. RESULTS: We identified 2353 men with prostate cancer and next generation somatic tumor sequencing evaluable for analysis in PROMISE, including 8% current, 39% former, and 52% never smokers. Current smokers were more likely to be younger and to have metastatic (M1 or N1) disease at diagnosis, and less likely to have prior local therapy (all p < 0.001). Current smoking was associated with worse OS from diagnosis (99.9 mo vs 137.6 mo, HR 1.42, 95% CI 1.14-1.77), which remained significant after adjusting for disease characteristics. We found no difference in the percentage of NEPC at initial diagnosis or at any time between current, former, and never smokers (p = 0.8). We found positive associations between smoking status and genetic alterations in SPOP (current: 15%, former 6.7%, never 3.8%; p = 0.018), FGFR1 (current 10%, former 0.4%, never 1.1% p = 0.001), and ARID1A (current 5.1%, former 2.2%, never 0.4%; p = 0.035) in patients with metastatic androgen pathway modulator sensitive prostate cancer (APMS). CONCLUSION: Active smoking is associated with worse overall and prostate cancer specific survival as compared to never/former smoking and was associated with specific tumor genetic alterations but not small cell/NEPC transformation.

Journal Article↗

PubMed2026

Improved Genomic Resources for the swordtail cricket, Laupala kohalensis Otte 1994.

Advances in genetic tools such as next and third generation sequencing, paired with a focus on representative clades, provide insight into how processes including adaptation, admixture, and genome structure shape the evolution and maintenance of species. However, our understanding of the genomics of speciation is dominated by systems where ecological adaptations are thought to cause initial barriers to gene exchange. In contrast to other model systems, the 38 species of the genus Laupala constitute a very rapid radiation, where evolution of reproductive barriers and speciation is thought to be driven by sexual selection. Here, with novel PacBio HiFi reads and RNA- and Iso-Seq data, we provide a highly contiguous, chromosome-level genome and markedly improved annotation of the endemic Hawaiian cricket, Laupala kohalensis Otte, 1994. Our new resources advance previous efforts, placing 99% of 47 scaffolds on 7 autosomes and 1 sex chromosome in the 1.67 Gb assembly, with a 98.8% BUSCO score (insecta_db10), N50 of ~268 Mb, and L50 of 3. Using a custom repeat library, we estimate the genome to have 46.09% repeat content, and the new annotation includes an increased estimate of 17,670 genes, which coincides with that known from other Orthopterans. Notably, we find a large nuclear DNA segment of mitochondrial origin on chromosome 7. This new resource provides a powerful tool to identify and compare genomic causes of phenotypic diversification in a system characterized by strong signatures of sexual differentiation, representing an underappreciated but potentially widespread cause of speciation.

Hawaii↗

PubMed1999

Clinical informatics: 2000 and beyond.

Healthcare has begun to flounder in the mounting flood of data available from automated monitoring equipment, microprocessor controlled life-support equipment, such as ventilators, ever more sophisticated laboratory tests, and the myriad of minor technological wonders that every hospital and clinic seem to collect. It is no longer enough to merely display the data in a large spreadsheet or on a complex, colorful time-sequence graph. The next generation of healthcare information systems must help the clinician to assimilate the myriad of data and to make fast and effective decisions. The following is a list of features that the next generation of computer systems will have to include if they are to have a significant impact on the quality of patient care: data acquisition, data storage, information display, data processing, and decision support. By automating or streamlining repetitive or complex tasks, correlating and presenting complex and potentially confusing data, and tracking patient outcomes, the computer can augment clinicians' skills to improve patient care.

Computer Systems↗

PubMed1991

High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs.

With the goal of developing techniques for DNA insertional mutagenesis in zebrafish, we established procedures for rapidly obtaining and injecting large numbers of fertilized eggs. Using either of two plasmid constructs, we injected uncut DNA into fertilized eggs at the one- or two-cell stage. Fish hatched from injected eggs were raised to sexual maturity, and the frequency of transgenic founder fish was determined by pair-mating the fish and testing DNA extracted from pools of their 16-hr-old offspring by the polymerase chain reaction (PCR) and then Southern analysis. Eggs injected with one of two different plasmids yielded no transgenic fish, but 7-25% (19 of 115 overall) of the eggs injected with the other plasmid transmitted the injected sequences to their offspring (F1). Of seven lines studied further, all were able to pass the foreign DNA sequences to the next (F2) generation. Inheritance in the F2 generation was Mendelian in the five lines tested. PCR and Southern analysis indicated that the plasmid sequences were present in multiple copies, probably tandemly arranged. Two founder fish carried more than one independent integration of the plasmid sequences. The line studied in more detail was a mosaic carrying two independently segregating copies of the transgene in one germ cell and a third copy in another germ-line precursor cell. The ability to obtain and inject large numbers of zebrafish eggs combined with a high frequency of germ-line integration may be steps toward the goal of being able to perform insertional mutagenesis with this organism.

Animals↗

PubMedDate not supplied

On graphical and numerical characterization of proteomics maps.

We outlined a mathematical approach suitable for characterization of experimental data given by 2-D densitograms. In particular we consider numerical characterization of proteomics maps. The basis of our approach is to order "spots" of a 2-D map and assign them unique labels (that in general will depend on the criteria used for ordering). In this way a map is "translated" into a sequence. In the next step one associates with the generated sequence a geometrical path and views such a path as a mathematical object that needs characterization. We have ordered spots representing proteins in 2-D gel plates according to their relative intensities which results in a zigzag path that produces a complicated "fingerprint" pattern. Mathematical characterization of zigzag pattern follows similar mathematical characterizations of embedded patterns based on matrices, the elements of which are given as quotients of Euclidean distance between spots and the distance along the zigzag path. The leading eigenvalue of constructed matrices is taken to represent characterization of the original 2-D map. Comparison of different 2-D maps (simulated by using random generator) allows one to construct partial order, which although qualitative in nature gives some insight into perturbation induced by foreign agents to the proteome of the control cell.

Computer Graphics↗

PubMed2002

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor.

High throughput DNA sequencing has been performed by using a microfabricated 96-channel radial capillary array electrophoresis (microCAE) microchannel plate detected by a 4-color rotary confocal fluorescence scanner. The microchannel plate features a novel injector for uniform sieving matrix loading as well as high resolution, tapered turns that provide an effective separation length of 15.9 cm on a compact 150-mm diameter wafer. Expanded common buffer chambers for the cathode, anode, and waste reservoirs are used to simplify electrode addressing and to counteract buffering capacity depletion arising from the high electrophoretic current. DNA sequencing data from 95 successful lanes out of 96 lanes run in parallel were batch-processed with basefinder, producing an average read length of 430 bp (phred q > or = 20). Phred quality values were found to exceed 40 (0.01% probability of incorrectly calling a base) for over 80% of the read length. The microCAE system demonstrated here produces sequencing data at a rate of 1.7 kbp/min, a 5-fold increase over current commercial capillary array electrophoresis technology. Additionally, this system permits lower reagent volumes and lower sample concentrations, and it presents numerous possibilities for integrated sample preparation and handling. The unique capabilities of microCAE technology should make it the next generation, high performance DNA sequencing platform.

Base Sequence↗

PubMed2025

Kaposi Sarcoma-Associated Herpesvirus Sequencing in People Living With HIV in the Southern United States Reveals Subtype Diversity and Multiple Infections.

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and lymphoproliferative diseases collectively identified as KSHV-associated diseases (KAD). While KAD incidence has decreased across the United States, regional and population-based variability exists, with higher rates in southern states. To understand the molecular epidemiology of KSHV in this region, samples were collected from people living with HIV (PWH) with or without history of KAD. PWH, mainly men who have sex with men (MSM), were recruited from a large, urban hospital system in Dallas, Texas, in two separate studies. The studies included 220 individuals without KAD and 59 patients with KAD. Whole blood and/or oral fluids were collected and tested by qPCR. KSHV subtypes were determined from 66 of 85 individuals with detectable KSHV loads by a combination of next-generation and targeted Sanger sequencing. All major KSHV subtypes, except D, were observed including subtypes E and F. In each of three individuals, multiple KSHV genome variants were identified. This study importantly highlights KSHV subtype diversity in the southern United States, which is an area with a high KS incidence. Genome diversity and multiple infections merit epidemiological consideration, including for the future development of vaccines.

Humans↗

PubMed2004

Simultaneous acquisition of multiple orders of intermolecular multiple-quantum coherence images in vivo.

Until recently, NMR imaging with intermolecular multiple-quantum coherences (iMQCs) has been based on the acquisition of a single echo. In vivo studies of iMQC image contrast would greatly benefit from a method that could acquire several orders of quantum coherence during the same acquisition. This would enable comparison of the image contrast for various orders and eliminate image coregistration problems between scans. It has previously been demonstrated that multiple orders of iMQC images can be simultaneously acquired of a simple phantom. Here, we examine the technique and its effect on biological tissue, both in vivo and in vitro. First, we establish the effectiveness of the iMQC sequence in vivo using earthworms as specimens. We then further show that the multi-CRAZED sequence enhances detection of next generation (nanoparticle) contrast agents on excised tumor tissue.

Animals↗

PubMed2005

Zebrafish to humans: evolution of the alpha3-chain of type IV collagen and emergence of the autoimmune epitopes associated with Goodpasture syndrome.

Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human alpha3(IV)NC1 domains bind autoantibodies. The alpha3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio alpha3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio alpha3(IV)NC1 domain. In contrast to recombinant human alpha3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio alpha3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human alpha3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years.

Animals↗

PubMed2004

Group-theoretic structure in the sequential flow of sensory experiences.

The hypothesis presented here is that in a given person, the overall configuration of sensory variables at any given moment, i.e. everything that the person is seeing, hearing, etc., can be treated as belonging to an infinite sequence of such momentary configurations, with the sequence being generated by a particular mathematical group. The ideas behind this are as follows. If a person's various moment-to-moment sensory experiences can all be described within the same coordinate system, then they can be treated as objects that sequentially transform into one another. If they are also encoded by brain events, then the person's own encounters with this encoding, e.g. when participating in brain-stimulation experiments, amount to instances where a given sensory experience inevitably follows other sensory experiences occurring in a specific sequence. For example, a person's visual encounters with certain equipment-settings might always be followed by a particular visual hallucination. If there exist sequences like this for any given momentary sensory experience, then these collectively form the person's sensory code, as it would be encountered from the person's own perspective. The person's ongoing flow of sensory experiences may then be treated as part of the infinite sequence implied by the various code-conserving ways in which sensory experiences can flow from one to the next. If this sequence can be generated by a finite number of code-conserving segments that transform into each other and link together end to end, then a person's full range of sensory experiences can be defined in group-theoretic terms.

Adaptation, Physiological↗

PubMed1993

The structure and function of the HSV DNA replication proteins: defining novel antiviral targets.

The absolute dependence of herpes simplex virus (HSV) replication on HSV DNA polymerase and six other viral-encoded replication proteins implies that specific inhibitors of these proteins' functions would be potent antiviral agents. The only currently licensed anti-herpes simplex drug, acyclovir, is an inhibitor of HSV DNA polymerase and is widely held to block viral replication primarily by specifically inhibiting viral DNA replication. In spite of the substantial advance in HSV therapy in recent years through the introduction of acyclovir, this anti-HSV compound and most of the other compounds under pharmaceutical development are substrate analogs. Since antiviral drug resistance has become an issue of increasing clinical importance, the need for structurally unrelated agents which incorporate novel mechanisms of viral inhibition is apparent. Understanding the structure and function of herpesvirus DNA polymerase and its interaction with the other six essential replication proteins at the replication origin should assist us in designing the next generation of therapeutic agents. The sequences of these proteins have been deduced and the proteins themselves have been expressed and purified in a variety of systems. The current challenge, therefore, is to use the available information about these proteins to identify and develop new, exquisitely specific antiviral therapeutics. In this review, we have summarized the current approaches and the results of structure/function studies of the herpes virus proteins essential for DNA replication, with the goal of more precisely defining novel antiviral targets.

Amino Acid Sequence↗

PubMed2003

Controlling the double helix.

Chromatin is the complex of DNA and proteins in which the genetic material is packaged inside the cells of organisms with nuclei. Chromatin structure is dynamic and exerts profound control over gene expression and other fundamental cellular processes. Changes in its structure can be inherited by the next generation, independent of the DNA sequence itself.

Animals↗

PubMed2004

Histone modification enzymes: novel targets for cancer drugs.

In eukaryotes, genomic DNA is packaged with histone proteins into the cell nucleus as chromatin, condensing the DNA > 10,000-fold. Chromatin is highly dynamic and exerts profound control on gene expression. Localised chromatin decondensation facilitates access of nuclear machinery. Chromatin displays epigenetic inheritance, in that changes in its structure can pass to the next generation independently of the DNA sequence itself. It is now clear that the post-translational modification of histones, for example, acetylation, methylation and phosphorylation, plays a crucial role in the regulation of nuclear function through the 'histone code'. There has been significant progress in identifying and understanding the enzymes that control these complex processes, in particular histone acetyltransferases and histone deacetylases. The exciting discovery that compounds inhibiting histone deacetylase activity also have antitumour properties has focused attention on their use as anticancer drugs. As a consequence, there is ongoing evaluation of several histone deacetylase inhibitor compounds in Phase I and II clinical trials with promising early results. It is likely that many of the enzymes involved in the control of histone modification will provide therapeutic opportunities for the treatment of cancer, including histone methyltransferases and Aurora kinases.

Acetylation↗

PubMed2005

Emerging technologies in DNA sequencing.

Demand for DNA sequence information has never been greater, yet current Sanger technology is too costly, time consuming, and labor intensive to meet this ongoing demand. Applications span numerous research interests, including sequence variation studies, comparative genomics and evolution, forensics, and diagnostic and applied therapeutics. Several emerging technologies show promise of delivering next-generation solutions for fast and affordable genome sequencing. In this review article, the DNA polymerase-dependent strategies of Sanger sequencing, single nucleotide addition, and cyclic reversible termination are discussed to highlight recent advances and potential challenges these technologies face in their development for ultrafast DNA sequencing.

Animals↗

PubMed1999

Stable-isotope-assisted MALDI-TOF mass spectrometry for accurate determination of nucleotide compositions of PCR products.

In parallel with a large-scale sequencing effort, the human genome project will need next-generation tools for accurate and efficient analyses of the enormous pool of DNA sequences. Such analyses are required for (a) validation of DNA sequences, (b) comparison of a parent (known) sequence with a related (unknown) sequence, and (c) characterization of sequence polymorphisms in various genes, especially those associated with genetically inherited human diseases. Here, we report a novel method that combines stable isotope 13C/15N labeling of PCR products of the target sequences with analysis of the mass shifts by mass spectrometry (MS). The mass shift due to the labeling of a single type of nucleotide (i.e., A, T, G, or C) reveals the number of that type of nucleotide in a given DNA fragment. Using this technique, we have accurately determined nucleotide compositions of DNA fragments. The method has also been applied to score a known single-nucleotide polymorphism (SNP). The comparisons of nucleotide compositions determined by our method among homologous sequences are useful in sequence validation, sequence comparison, and characterizations of sequence polymorphisms.

Cadherins↗