A trimeric DNA polymerase complex increases the native replication processivity

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase–polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Home and Online Management and Evaluation of Blood Pressure (HOME BP) using a digital intervention in poorly controlled hypertension: randomised controlled trial

Objective: The HOME BP (Home and Online Management and Evaluation of Blood Pressure) trial aimed to test a digital intervention for hypertension management in primary care by combining self-monitoring of blood pressure with guided self-management. Design: Unmasked randomised controlled trial with automated ascertainment of primary endpoint. Setting: 76 general practices in the United Kingdom. Participants: 622 people with treated but poorly controlled hypertension (>140/90 mm Hg) and access to the internet. Interventions: Participants were randomised by using a minimisation algorithm to self-monitoring of blood pressure with a digital intervention (305 participants) or usual care (routine hypertension care, with appointments and drug changes made at the discretion of the general practitioner; 317 participants). The digital intervention provided feedback of blood pressure results to patients and professionals with optional lifestyle advice and motivational support. Target blood pressure for hypertension, diabetes, and people aged 80 or older followed UK national guidelines. Main outcome measures: The primary outcome was the difference in systolic blood pressure (mean of second and third readings) after one year, adjusted for baseline blood pressure, blood pressure target, age, and practice, with multiple imputation for missing values. Results: After one year, data were available from 552 participants (88.6%) with imputation for the remaining 70 participants (11.4%). Mean blood pressure dropped from 151.7/86.4 to 138.4/80.2 mm Hg in the intervention group and from 151.6/85.3 to 141.8/79.8 mm Hg in the usual care group, giving a mean difference in systolic blood pressure of −3.4 mm Hg (95% confidence interval −6.1 to −0.8 mm Hg) and a mean difference in diastolic blood pressure of −0.5 mm Hg (−1.9 to 0.9 mm Hg). Results were comparable in the complete case analysis and adverse effects were similar between groups. Within trial costs showed an incremental cost effectiveness ratio of £11 ($15, €12; 95% confidence interval £6 to £29) per mm Hg reduction. Conclusions: The HOME BP digital intervention for the management of hypertension by using self-monitored blood pressure led to better control of systolic blood pressure after one year than usual care, with low incremental costs. Implementation in primary care will require integration into clinical workflows and consideration of people who are digitally excluded. Trial registration: ISRCTN13790648

My46: a Web-based tool for self-guided management of genomic test results in research and clinical settings.

A major challenge to implementing precision medicine is the need for an efficient and cost-effective strategy for returning individual genomic test results that is easily scalable and can be incorporated into multiple models of clinical practice. My46 is a Web-based tool for managing the return of genetic results that was designed and developed to support a wide range of approaches to disclosing results, ranging from traditional face-to-face disclosure to self-guided models. My46 has five key functions: set and modify results-return preferences, return results, educate, manage the return of results, and assess the return of results. These key functions are supported by six distinct modules and a suite of features that enhance the user experience, ease site navigation, facilitate knowledge sharing, and enable results-return tracking. My46 is a potentially effective solution for returning results and supports current trends toward shared decision making between patients and providers and patient-driven health management.Genet Med 19 4, 467-475

Suppressive subtractive hybridization analysis of Rhipicephalus (Boophilus) microplus larval and adult transcript expression during attachment and feeding

Ticks, as blood-feeding ectoparasites, affect their hosts both directly and as vectors of viral, bacterial and protozoal diseases. The tick's mode of feeding means it must maintain intimate contact with the host in the face of host defensive responses for a prolonged time. The parasite–host interactions are characterized by the host response and parasite counter-response which result in a highly complex biological system that is barely understood. We conducted transcriptomic analyses utilizing suppressive subtractive hybridization (SSH) to identify transcripts associated with host attachment and feeding of larval, adult female and adult male ticks. Five SSH libraries resulted in 511 clones (assembled into 36 contigs and 90 singletons) from differentially expressed transcripts isolated from unattached frustrated larvae (95), feeding larvae (159), unattached frustrated adult female ticks (68), feeding adult female ticks (95) and male adult ticks (94 clones). Unattached ‘frustrated’ ticks were held in fabric bags affixed to cattle for up to 24 h to identify genes up-regulated prior to host penetration. Sequence analysis was based on BLAST, Panther, KOG and domain (CDD) analyses to assign functional groups for proteins including: cuticle proteins, enzymes (ATPases), ligand binding (histamine binding), molecular chaperone (prefoldin), nucleic acid binding (ribosomal proteins), putative salivary proteins, serine proteases, stress response (heat shock, glycine rich) and transporters. An additional 63% of all contigs and singletons were novel R. microplus transcripts or predicted proteins of unknown function. Expression was confirmed using quantitative real time PCR analysis of selected transcripts. This is the first comprehensive analysis of the R. microplus transcriptome from multiple stages of ticks and assists to elucidate the molecular events during tick attachment and development

Connectionist psycholinguistics : capturing the empirical data

Connectionist psycholinguistics is an emerging approach to modeling empirical data on human language processing using connectionist computational architectures. For almost 20 years, connectionist models have increasingly been used to model empirical data across many areas of language processing. We critically review four key areas: speech processing, sentence processing, language production, and reading aloud, and evaluate progress against three criteria: data contact, task veridicality, and input representativeness. Recent connectionist modeling efforts have made considerable headway toward meeting these criteria, although it is by no means clear whether connectionist (or symbolic) psycholinguistics will eventually provide an integrated model of full-scale human language processing

DNA Polymerases Drive DNA Sequencing-by-Synthesis Technologies: Both Past and Present

Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. E. coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger's dideoxy chain terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ⱷ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ⱷ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies