Kinase deoxyribozymes

Nature has developed the use of proteins and RNA as enzymes, while DNA is used for the storage and transfer of genetic information. Proteins and RNA are biopolymers that can fold into specific secondary and tertiary structures to enable catalysis. Considering the structural similarity to RNA, single-stranded DNA should also be able to form complex structures capable of catalyzing reactions. DNA catalysts have not been identified in nature, but in vitro selection has led to the identification of DNA catalysts for a variety of chemical reactions. Identification of new catalysts favors the use of DNA for multiple reasons. Amplification of functional DNA sequences is directly possible using natural polymerases, whereas amplification of RNA requires an additional reverse transcription step and amplification of proteins is not possible. The total number of possible sequences is smaller for nucleic acids (4n, where n is the number of residues) than for proteins (20n). Within this sequence space a large number of random nucleic acid sequences will fold into secondary and tertiary structures unlike proteins which require specific amino acid sequences to form complex structures. Therefore, in vitro selection experiments to identify DNA catalysts will cover a large portion of sequence space, and a large fraction of the covered space will contain structured DNA sequences with the potential to be catalytically active. The ease of synthesis and stability of DNA compared to RNA or proteins also provides an advantage for its use as a catalyst. Natural post-translational modifications (PTMs) are important in biological systems. PTMs modulate protein activity resulting in rapid changes to cellular processes. Studying the role of specific PTMs is often limited to the ability to generate site-specific post-translationally modified proteins of interest. Phosphorylation of amino acid side chains is an abundant natural PTM that is essential for cellular function. Protein kinases, which catalyze phosphorylation, are often motif specific. Engineering these natural kinases to change motif requirements is challenging and often results in decreased substrate specificity. To identify new catalysts for the site-specific phosphorylation of a desired protein the use of DNA as a catalytic biomolecule is advantageous because an initially random population of DNA sequences does not have substrate biases, and DNA is a large biopolymer with the possibility to interact specifically with the substrates. Both ribozymes and deoxyribozymes have been identified to catalyze the phosphorylation of oligonucleotides. However, previous efforts to identify kinase deoxyribozymes to catalyze the phosphorylation of amino acid side chains were unsuccessful because the -thiophosphoryl donor used was not stable in the selection conditions. As described herein, a new in vitro selection method was developed using a previously identified deoxyribozyme to separate the active deoxyribozymes from the inactive DNA sequences. This method led to the identification of the first kinase deoxyribozymes capable of phosphorylating tyrosine residues within a tethered peptide substrate using a bound 5′-triphosphorylated RNA oligonucleotide as the phosphoryl donor. Separate selection experiments were performed using 1 mM GTP as the phosphoryl donor. The identified DNA catalysts are able to phosphorylate tyrosine within a peptide substrate and require only low micromolar concentrations of GTP. Site-specific modification of proteins is often desired. Most deoxyribozymes identified to modify peptide substrates have been identified using peptide substrates containing the reactive residue flanked by alanine residues. Peptide sequences derived from natural proteins contain a variety of amino acid residues with diverse functional groups that could be a point of interaction between the peptide substrate and DNA catalyst. Selection experiments were performed with biologically derived peptide sequences to identify tyrosine kinase deoxyribozymes with the ability to phosphorylate peptides sequence-specifically. Of the three peptide substrates evaluated the use of one led to deoxyribozymes that are peptide motif-specific, the second peptide led to deoxyribozymes with partial peptide sequence-selectivity, and the third did not lead to the identification of deoxyribozymes. The identification of peptide motif-specific deoxyribozymes demonstrates that DNA catalysts can interact specifically with peptide substrates, and individual DNA enzymes can interact with the same peptide substrate in a different manner. The ability to phosphorylate substrates that are free in solution is desired. However, previously identified kinase deoxyribozymes are unable to phosphorylate untethered peptide substrates. Original efforts increased the length of the tether between the peptide substrate and DNA anchor to mimic a peptide free in solution. These selection experiments did not lead to deoxyribozymes, and further analysis of other deoxyribozymes with untethered peptide reactivity suggests the long tethers may interfere with catalysis. Further efforts have focused on the incorporation of hydrophobic modifications into the DNA catalysts to improve peptide binding. DNA aptamers containing hydrophobic modifications have improved protein binding. Increased binding affinity between the peptide substrate and DNA catalyst may enable untethered peptide reactivity. While initial efforts focused on tyrosine phosphorylation, serine phosphorylation is also abundant in nature. Serine kinase deoxyribozymes have been identified to phosphorylate serine within tightly tethered peptide substrates using 5′-triphosphorylated RNA as the phosphoryl donor. Subsequent efforts to increase the tether length or use ATP as the phosphoryl donor were unsuccessful. Efforts to improve DNA catalysts with the ability to phosphorylate serine include using biologically derived peptide sequences to increase interactions between the deoxyribozyme and peptide substrate, and incorporating catalytically participatory modifications into the DNA enzymes

Recipient screening in IVF: First data from women undergoing anonymous oocyte donation in Dublin

BACKGROUND: Guidelines for safe gamete donation have emphasised donor screening, although none exist specifically for testing oocyte recipients. Pre-treatment assessment of anonymous donor oocyte IVF treatment in Ireland must comply with the European Union Tissues and Cells Directive (Directive 2004/23/EC). To determine the effectiveness of this Directive when applied to anonymous oocyte recipients in IVF, we reviewed data derived from selected screening tests performed in this clinical setting. METHODS: Data from tests conducted at baseline for all women enrolling as recipients (n = 225) in the anonymous oocyte donor IVF programme at an urban IVF referral centre during a 24-month period were analysed. Patient age at programme entry and clinical pregnancy rate were also tabulated. All recipients had at least one prior negative test for HIV, Hepatitis B/C, chlamydia, gonorrhoea and syphilis performed by her GP or other primary care provider before reproductive endocrinology consultation. RESULTS: Mean (±SD) age for donor egg IVF recipients was 40.7 ± 4.2 yrs. No baseline positive chlamydia, gonorrhoea or syphilis screening results were identified among recipients for anonymous oocyte donation IVF during the assessment interval. Mean pregnancy rate (per embryo transfer) in this group was 50.5%. CONCLUSION: When tests for HIV, Hepatitis B/C, chlamydia, gonorrhoea and syphilis already have been confirmed to be negative before starting the anonymous donor oocyte IVF sequence, additional (repeat) testing on the recipient contributes no new clinical information that would influence treatment in this setting. Patient safety does not appear to be enhanced by application of Directive 2004/23/EC to recipients of anonymous donor oocyte IVF treatment. Given the absence of evidence to quantify risk, this practice is difficult to justify when applied to this low-risk population

The Journal of BSN Honors Research, Volume 7, Issue 1, Summer 2014

Papers submitted to the University of Kansas School of Nursing in partial fulfillment of the requirements for the Nursing Honors Program.The University of Kansas School of Nursing Bachelor of Science Nursing Honors Progra

The Wide-field Infrared Survey Explorer (WISE): Mission Description and Initial On-orbit Performance

The all sky surveys done by the Palomar Observatory Schmidt, the European Southern Observatory Schmidt, and the United Kingdom Schmidt, the InfraRed Astronomical Satellite and the 2 Micron All Sky Survey have proven to be extremely useful tools for astronomy with value that lasts for decades. The Wide-field Infrared Survey Explorer is mapping the whole sky following its launch on 14 December 2009. WISE began surveying the sky on 14 Jan 2010 and completed its first full coverage of the sky on July 17. The survey will continue to cover the sky a second time until the cryogen is exhausted (anticipated in November 2010). WISE is achieving 5 sigma point source sensitivities better than 0.08, 0.11, 1 and 6 mJy in unconfused regions on the ecliptic in bands centered at wavelengths of 3.4, 4.6, 12 and 22 microns. Sensitivity improves toward the ecliptic poles due to denser coverage and lower zodiacal background. The angular resolution is 6.1, 6.4, 6.5 and 12.0 arc-seconds at 3.4, 4.6, 12 and 22 microns, and the astrometric precision for high SNR sources is better than 0.15 arc-seconds.Comment: 22 pages with 19 included figures. Updated to better match the accepted version in the A

Routes for breaching and protecting genetic privacy

We are entering the era of ubiquitous genetic information for research, clinical care, and personal curiosity. Sharing these datasets is vital for rapid progress in understanding the genetic basis of human diseases. However, one growing concern is the ability to protect the genetic privacy of the data originators. Here, we technically map threats to genetic privacy and discuss potential mitigation strategies for privacy-preserving dissemination of genetic data.Comment: Draft for comment

Caribbean Corals in Crisis: Record Thermal Stress, Bleaching, and Mortality in 2005

BACKGROUND The rising temperature of the world's oceans has become a major threat to coral reefs globally as the severity and frequency of mass coral bleaching and mortality events increase. In 2005, high ocean temperatures in the tropical Atlantic and Caribbean resulted in the most severe bleaching event ever recorded in the basin. METHODOLOGY/PRINCIPAL FINDINGS Satellite-based tools provided warnings for coral reef managers and scientists, guiding both the timing and location of researchers' field observations as anomalously warm conditions developed and spread across the greater Caribbean region from June to October 2005. Field surveys of bleaching and mortality exceeded prior efforts in detail and extent, and provided a new standard for documenting the effects of bleaching and for testing nowcast and forecast products. Collaborators from 22 countries undertook the most comprehensive documentation of basin-scale bleaching to date and found that over 80% of corals bleached and over 40% died at many sites. The most severe bleaching coincided with waters nearest a western Atlantic warm pool that was centered off the northern end of the Lesser Antilles. CONCLUSIONS/SIGNIFICANCE Thermal stress during the 2005 event exceeded any observed from the Caribbean in the prior 20 years, and regionally-averaged temperatures were the warmest in over 150 years. Comparison of satellite data against field surveys demonstrated a significant predictive relationship between accumulated heat stress (measured using NOAA Coral Reef Watch's Degree Heating Weeks) and bleaching intensity. This severe, widespread bleaching and mortality will undoubtedly have long-term consequences for reef ecosystems and suggests a troubled future for tropical marine ecosystems under a warming climate.This work was partially supported by salaries from the NOAA Coral Reef Conservation Program to the NOAA Coral Reef Conservation Program authors. NOAA provided funding to Caribbean ReefCheck investigators to undertake surveys of bleaching and mortality. Otherwise, no funding from outside authors' institutions was necessary for the undertaking of this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

An exploration of the family resilience needs of a rural community in South Africa: a sequential explanatory mixed methodological study design

The aim of the study is to identify and explore family resilience needs in a rural community in the West Coast region of South Africa. An explanatory mixed methodological sequential design was implemented. Firstly, Sixbey’s (2005) Family Resilience Assessment Scale, was employed to conduct the quantitative assessment via a door-to-door sample of convenience identified with the assistance of a local nongovernmental organisation. Of the 656 participants, 39.8% were male and 60.2% were female, with an average age of 37.90 years (standard deviation 13.92). Secondly, four focus groups involving 27 community participants provided qualitative data. Results from the quantitative assessment show that family connectedness and utilising social and economic resources were the lowest scoring, and belief systems the highest scoring, dimensions in family resilience. Based on the quantitative findings and the discussions, three thematic categories emerged: community and family challenges; community belief systems; and current family functioning and organisational patterns. A number of families and groups within the community were able to provide feedback, recommendations and work collaboratively in this study. This contributed to the argument we make for the transformative mixed methods paradigm that is discussed. This study provides further insight into the theory of family resilience.ISI & Scopu

Effect of Depth and Duration of Cooling on Death or Disability at Age 18 Months Among Neonates With Hypoxic-Ischemic Encephalopathy: A Randomized Clinical Trial

Importance Hypothermia for 72 hours at 33.5°C for neonatal hypoxic-ischemic encephalopathy reduces death or disability, but rates continue to be high. Objective To determine if cooling for 120 hours or to a temperature of 32.0°C reduces death or disability at age 18 months in infants with hypoxic-ischemic encephalopathy. Design, Setting, and Participants Randomized 2 × 2 factorial clinical trial in neonates (≥36 weeks’ gestation) with hypoxic-ischemic encephalopathy at 18 US centers in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network between October 2010 and January 2016. Interventions A total of 364 neonates were randomly assigned to 4 hypothermia groups: 33.5°C for 72 hours (n = 95), 32.0°C for 72 hours (n = 90), 33.5°C for 120 hours (n = 96), or 32.0°C for 120 hours (n = 83). Main Outcomes and Measures The primary outcome was death or moderate or severe disability at 18 to 22 months of age adjusted for center and level of encephalopathy. Severe disability included any of Bayley Scales of Infant Development III cognitive score less than 70, Gross Motor Function Classification System (GMFCS) level of 3 to 5, or blindness or hearing loss despite amplification. Moderate disability was defined as a cognitive score of 70 to 84 and either GMFCS level 2, active seizures, or hearing with amplification. Results The trial was stopped for safety and futility in November 2013 after 364 of the planned 726 infants were enrolled. Among 347 infants (95%) with primary outcome data (mean age at follow-up, 20.7 [SD, 3.5] months; 42% female), death or disability occurred in 56 of 176 (31.8%) cooled for 72 hours and 54 of 171 (31.6%) cooled for 120 hours (adjusted risk ratio, 0.92 [95% CI, 0.68-1.25]; adjusted absolute risk difference, −1.0% [95% CI, −10.2% to 8.1%]) and in 59 of 185 (31.9%) cooled to 33.5°C and 51 of 162 (31.5%) cooled to 32.0°C (adjusted risk ratio, 0.92 [95% CI, 0.68-1.26]; adjusted absolute risk difference, −3.1% [95% CI, −12.3% to 6.1%]). A significant interaction between longer and deeper cooling was observed (P = .048), with primary outcome rates of 29.3% at 33.5°C for 72 hours, 34.5% at 32.0°C for 72 hours, 34.4% at 33.5°C for 120 hours, and 28.2% at 32.0°C for 120 hours. Conclusions and Relevance Among term neonates with moderate or severe hypoxic-ischemic encephalopathy, cooling for longer than 72 hours, cooling to lower than 33.5°C, or both did not reduce death or moderate or severe disability at 18 months of age. However, the trial may be underpowered, and an interaction was found between longer and deeper cooling. These results support the current regimen of cooling for 72 hours at 33.5°C

Genome-wide association of familial prostate cancer cases identifies evidence for a rare segregating haplotype at 8q24.21

Previous genome-wide association studies (GWAS) of prostate cancer risk focused on cases unselected for family history and have reported over 100 significant associations. The International Consortium for Prostate Cancer Genetics (ICPCG) has now performed a GWAS of 2511 (unrelated) familial prostate cancer cases and 1382 unaffected controls from 12 member sites. All samples were genotyped on the Illumina 5M+exome single nucleotide polymorphism (SNP) platform. The GWAS identified a significant evidence for association for SNPs in six regions previously associated with prostate cancer in population-based cohorts, including 3q26.2, 6q25.3, 8q24.21, 10q11.23, 11q13.3, and 17q12. Of note, SNP rs138042437 (p = 1.7e−8) at 8q24.21 achieved a large estimated effect size in this cohort (odds ratio = 13.3). 116 previously sampled affected relatives of 62 risk-allele carriers from the GWAS cohort were genotyped for this SNP, identifying 78 additional affected carriers in 62 pedigrees. A test for an excess number of affected carriers among relatives exhibited strong evidence for co-segregation of the variant with disease (p = 8.5e−11). The majority (92 %) of risk-allele carriers at rs138042437 had a consistent estimated haplotype spanning approximately 100 kb of 8q24.21 that contained the minor alleles of three rare SNPs (dosage minor allele frequencies <1.7 %), rs183373024 (PRNCR1), previously associated SNP rs188140481, and rs138042437 (CASC19). Strong evidence for co-segregation of a SNP on the haplotype further characterizes the haplotype as a prostate cancer pre-disposition locus

Effect of Therapeutic Hypothermia Initiated After 6 Hours of Age on Death or Disability Among Newborns With Hypoxic-Ischemic Encephalopathy: A Randomized Clinical Trial

Importance: Hypothermia initiated at less than 6 hours after birth reduces death or disability for infants with hypoxic-ischemic encephalopathy at 36 weeks' or later gestation. To our knowledge, hypothermia trials have not been performed in infants presenting after 6 hours. Objective: To estimate the probability that hypothermia initiated at 6 to 24 hours after birth reduces the risk of death or disability at 18 months among infants with hypoxic-ischemic encephalopathy. Design, Setting, and Participants: A randomized clinical trial was conducted between April 2008 and June 2016 among infants at 36 weeks' or later gestation with moderate or severe hypoxic-ischemic encephalopathy enrolled at 6 to 24 hours after birth. Twenty-one US Neonatal Research Network centers participated. Bayesian analyses were prespecified given the anticipated limited sample size. Interventions: Targeted esophageal temperature was used in 168 infants. Eighty-three hypothermic infants were maintained at 33.5°C (acceptable range, 33°C-34°C) for 96 hours and then rewarmed. Eighty-five noncooled infants were maintained at 37.0°C (acceptable range, 36.5°C-37.3°C). Main Outcomes and Measures: The composite of death or disability (moderate or severe) at 18 to 22 months adjusted for level of encephalopathy and age at randomization. Results: Hypothermic and noncooled infants were term (mean [SD], 39 [2] and 39 [1] weeks' gestation, respectively), and 47 of 83 (57%) and 55 of 85 (65%) were male, respectively. Both groups were acidemic at birth, predominantly transferred to the treating center with moderate encephalopathy, and were randomized at a mean (SD) of 16 (5) and 15 (5) hours for hypothermic and noncooled groups, respectively. The primary outcome occurred in 19 of 78 hypothermic infants (24.4%) and 22 of 79 noncooled infants (27.9%) (absolute difference, 3.5%; 95% CI, -1% to 17%). Bayesian analysis using a neutral prior indicated a 76% posterior probability of reduced death or disability with hypothermia relative to the noncooled group (adjusted posterior risk ratio, 0.86; 95% credible interval, 0.58-1.29). The probability that death or disability in cooled infants was at least 1%, 2%, or 3% less than noncooled infants was 71%, 64%, and 56%, respectively. Conclusions and Relevance: Among term infants with hypoxic-ischemic encephalopathy, hypothermia initiated at 6 to 24 hours after birth compared with noncooling resulted in a 76% probability of any reduction in death or disability, and a 64% probability of at least 2% less death or disability at 18 to 22 months. Hypothermia initiated at 6 to 24 hours after birth may have benefit but there is uncertainty in its effectiveness