Structural and functional dissection of the vaccinia virus thymidine kinase enzyme

Thymidine kinase is a key enzyme in the nucleotide salvage pathway, catalyzing the production of dTMP from thymidine and ATP. In order to identify the structural features of the TK protein and/or primary amino acid sequences which contribute to the catalytic and regulatory activities of this enzyme, an in vitro transcription and translation system has been used in concert with protein engineering techniques for the production and phenotypic characterization of mutant and wild-type TK enzymes. Because of discrepancies in the literature regarding the quaternary structure of the VVTK, the native molecular weight and quaternary structure was determined to be an 80kDa homotetrameric enzyme by glycerol gradient fractionation, gel filtration and glutaraldehyde cross-linking analyses. Computer-assisted alignment of the predicted amino acid sequences derived from cellular and poxvirus TK genes identified seven highly-conserved domains distributed throughout the VVTK polypeptide (domains I-VII). Domain I (amino acid residues 11-18 ) exhibits a high degree of similarity to both ATP and GTP binding site consensus sequences, although the VVTK utilizes only ATP as a phosphate donor. Site directed mutagenesis and ATP-agarose affinity chromatography techniques were employed to confirm that this region was responsible for ATP binding and to determine which amino acids were essential for efficient binding. The TK gene (tdk) from E. coli was isolated and sequenced to serve as a prokaryotic enzyme with which to compare VVTK. The alignment revealed only 23% shared identity with VVTK and, interestingly, the identical and similar residues were clustered into three of the seven domains identified previously (domains I, III and VII). Preliminary evidence supports domain III (residues 78-90) as a putative magnesium binding site and that a highly conserved cysteine residue (cysteine 170) within domain VII (residues 168-171) may be a component of the catalytic site. Secondary structure alignment between Herpes Simplex Virus (HSV) TK and monkeypox TK (a close relative of VVTK) revealed that the putative nucleoside binding site of HSVTK aligns with residues within domain IV. Replacement of a VVTK residue (Q114) with the corresponding residue of HSVTK (an aspartic acid) greatly alters the substrate specificity and dTTP sensitivity of VVTK

The Role of Herpes Simplex Virus-1 Thymidine Kinase Alanine 168 in Substrate Specificity

Herpes simplex virus type 1 (HSV) thymidine kinase (TK) has been widely used in suicide gene therapy for the treatment of cancer due to its broad substrate specificity and the inability of the endogenous human TK to phosphorylate guanosine analogs such as ganciclovir (GCV). The basis of suicide gene therapy is the introduction of a gene that encodes a prodrug-activating enzyme into tumor cells. After administration, the prodrug is selectively converted to a toxic drug by the suicide gene product thereby bringing about the eradication of the cancer cells. A major drawback to this therapy is the low activity the enzyme displays towards the prodrugs, requiring high prodrug doses that result in adverse side effects. Earlier studies revealed two HSV TK variants (SR39 and mutant 30) derived by random mutagenesis with enhanced activities towards GCV in vitro and in vivo. While these mutants contain multiple amino acid substitutions, molecular modeling suggests that substitutions at alanine 168 (A168) may be responsible for the observed increase in prodrug sensitivity. To evaluate this, site-directed mutagenesis was used to individually substitute A168 with phenylalanine or tyrosine to reflect the mutations found in SR39 and mutant 30, respectively. Additionally, kinetic parameters and the ability of these mutants to sensitize tumor cells to GCV in comparison to wild-type thymidine kinase were determined

The 1.14 Å Crystal Structure of Yeast Cytosine Deaminase Evolution of Nucleotide Salvage Enzymes and Implications for Genetic Chemotherapy

AbstractCytosine deaminase (CD) catalyzes the deamination of cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway. The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 Å resolution, respectively. The enzyme forms an α/β fold similar to bacterial cytidine deaminase, but with no similarity to the α/β barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase. The structures observed for bacterial, fungal, and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates

Impact Of Tactile-Cued Self-Monitoring On Independent Biology Work For Secondary Students With Attention Deficit Hyperactivity Disorder

Results from a multiple baseline with changing conditions design across high school students with Attention Deficit Hyperactivity Disorder (ADHD) indicated that the students increased the percentage of independent work they completed in their general education biology class after learning tactile-cued self-monitoring. Students maintained high percentages for completed work when the rate of tactile cues was faded from 1 per minute to 1 every 5 minutes, as well as when all tactile cues were withdrawn during a short-term maintenance phase.  Moreover, the students increased their correctly completed work from percentages substantially lower than the mean of their classmates to percentages that matched and surpassed the mean of their classmates. Qualitative data indicated that the participants and their co-teachers approved of the tactile-cued self-monitoring procedures. Results confirm and extend prior research findings that students improve performance during independent tasks after learning how to use tactile-cued self-monitoring and that students maintain improvements when the tactile cues are systematically faded. Although this research was conducted in a secondary school setting, the method also could be applied to higher education. Postsecondary disability resource center personnel might consider MotivAider use for students with ADHD and other disabilities that affect the capacity to stay on task

Revised National Pressure Ulcer Advisory Panel Pressure Injury Staging System: Revised Pressure Injury Staging System

Our understanding of pressure injury etiology and development has grown in recent years through research, clinical expertise, and global interdisciplinary expert collaboration. Therefore, the National Pressure Ulcer Advisory Panel (NPUAP) has revised the definition and stages of pressure injury. The revision was undertaken to incorporate the current understanding of the etiology of pressure injuries, as well as to clarify the anatomical features present or absent in each stage of injury. An NPUAP-appointed Task Force reviewed the literature and created drafts of definitions, which were then reviewed by stakeholders and the public, including clinicians, educators, and researchers around the world. Using a consensus-building methodology, these revised definitions were the focus of a multidisciplinary consensus conference held in April 2016. As a result of stakeholder and public input, along with the consensus conference, important changes were made and incorporated into the new staging definitions. The revised staging system uses the term injury instead of ulcer and denotes stages using Arabic numerals rather than Roman numerals. The revised definition of a pressure injury now describes the injuries as usually occurring over a bony prominence or under a medical or other device. The revised definition of a Stage 2 pressure injury seeks to clarify the difference between moisture-associated skin damage and injury caused by pressure and/or shear. The term suspected has been removed from the Deep Tissue Pressure Injury diagnostic label. Each definition now describes the extent of tissue loss present and the anatomical features that may or may not be present in the stage of injury. These important revisions reflect the methodical and collaborative approach used to examine the available evidence and incorporate current interdisciplinary clinical expertise into better defining the important phenomenon of pressure injury etiology and development

Directing diarrhoeal disease research towards disease-burden reduction

Despite gains in controlling mortality relating to diarrhoeal disease, the burden of disease remains unacceptably high. To refocus health research to target disease-burden reduction as the goal of research in child health, the Child Health and Nutrition Research Initiative developed a systematic strategy to rank health research options. This priority-setting exercise included listing of 46 competitive research options in diarrhoeal disease and their critical and quantitative appraisal by 10 experts based on five criteria for research that reflect the ability of the research to be translated into interventions and achieved disease-burden reduction. These criteria included the answerability of the research questions, the efficacy and effectiveness of the intervention resulting from the research, the maximal potential for disease-burden reduction of the interventions derived from the research, the affordability, deliverability, and sustainability of the intervention supported by the research, and the overall effect of the research-derived intervention on equity. Experts scored each research option independently to delineate the best investments for diarrhoeal disease control in the developing world to reduce the burden of disease by 2015. Priority scores obtained for health policy and systems research obtained eight of the top 10 rankings in overall scores, indicating that current investments in health research are significantly different from those estimated to be the most effective in reducing the global burden of diarrhoeal disease by 2015

Effects of Child and Maternal Histo-Blood Group Antigen Status on Symptomatic and Asymptomatic Enteric Infections in Early Childhood

Funding Information: Financial support. This work was funded by the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project (MAL-ED) is carried out as a collaborative project funded by the Bill & Melinda Gates Foundation (BMGF) (BMGF-47075), the Foundation for the National Institutes of Health, and the National Institutes of Health, Fogarty International Center, whereas additional support was obtained from BMGF for the examination of host innate factors on enteric disease risk and enteropathy (Grants OPP1066146 and OPP1152146; to M. N. K.). Additional funding was obtained from teh Sherrilyn and Ken Fisher Center for Environmental Infectious Diseases, Johns Hopkins School of Medicine (to M. N. K) and the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institues of health 1UL1TR001079. Acknowledgments. We thank the participants, their families, and the study community for their dedicated time and effort to better the understanding the transmission and more enduring impact of enteric infections in early childhood. We also thank the following: Jan Vinje (Centers for Disease Control and Prevention) for critical input and manuscript review; Dr. Leah Jager for consultation regarding the statistical analysis; Dr. Ben Jann (University of Bern, Switzerland) for guidance in generating the figures; Christine Szymanski for insight and encouragement, particularly regarding Campylobacter infection and disease patency; Chris Damman and Anita Zaidi for input on early iterations of the analysis; and Dick Guerrant for final reflections.Peer reviewe

A multi-stage genome-wide association study of bladder cancer identifies multiple susceptibility loci.

We conducted a multi-stage, genome-wide association study of bladder cancer with a primary scan of 591,637 SNPs in 3,532 affected individuals (cases) and 5,120 controls of European descent from five studies followed by a replication strategy, which included 8,382 cases and 48,275 controls from 16 studies. In a combined analysis, we identified three new regions associated with bladder cancer on chromosomes 22q13.1, 19q12 and 2q37.1: rs1014971, (P = 8 × 10⁻¹²) maps to a non-genic region of chromosome 22q13.1, rs8102137 (P = 2 × 10⁻¹¹) on 19q12 maps to CCNE1 and rs11892031 (P = 1 × 10⁻⁷) maps to the UGT1A cluster on 2q37.1. We confirmed four previously identified genome-wide associations on chromosomes 3q28, 4p16.3, 8q24.21 and 8q24.3, validated previous candidate associations for the GSTM1 deletion (P = 4 × 10⁻¹¹) and a tag SNP for NAT2 acetylation status (P = 4 × 10⁻¹¹), and found interactions with smoking in both regions. Our findings on common variants associated with bladder cancer risk should provide new insights into the mechanisms of carcinogenesis