24 results on '"Freeman AD"'
Search Results
2. Oral health in Australian HIV patients since the advent of combination antiretroviral therapy
- Author
-
Freeman, AD, primary, Liberali, SA, additional, Coates, EA, additional, and Logan, RM, additional
- Published
- 2012
- Full Text
- View/download PDF
3. The middle layer of lumbar fascia can transmit tensile forces capable of fracturing the lumbar transverse processes: an experimental study.
- Author
-
Barker PJ, Freeman AD, Urquhart DM, Anderson CR, and Briggs CA
- Abstract
BACKGROUND: Transversus abdominis and its aponeurotic attachment to the lumbar transverse processes via the middle layer of lumbar fascia are of proposed clinical and biomechanical importance. Moderate traction on these structures (simulating submaximal contraction of transversus abdominis) is reported to influence segmental motion, but their tensile capacity is unknown and the effects of sudden, maximal traction on these attachments and the transverse processes are uncertain. METHODS: In 15 embalmed cadaver abdomens, the middle layer of lumbar fascia was isolated, gripped and rapid tension applied in either a lateral or posteroanterior direction (simulating forces that may produce avulsion and traumatic fractures). Peak forces prior to tissue failure were recorded and the gross effects of traction documented. FINDINGS: Lumbar transverse process fractures were produced in all specimens; by transverse traction in 50% of tests and posteroanterior force in 80%. In the remainder the middle layer of lumbar fascia was torn. Mean transverse and posteroanterior peak forces reached in the middle layer of lumbar fascia prior to failure were 82 N (range 20-190 N) and 47 N (range 25-70 N), respectively. INTERPRETATION: The middle layer of lumbar fascia can transmit substantial tensile forces to lumbar vertebrae, capable of transverse process fracture under experimental conditions. Tensile capacity is likely to be even greater in-vivo. This suggests transversus abdominis and the middle layer of lumbar fascia can strongly influence vertebral motion, should be incorporated in biomechanical models of the spine and considered as potential contributors to transverse process fractures by avulsion. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
4. Oral conditions and their social impact among HIV dental patients, 18 years on
- Author
-
SA Liberali, Lisa Jamieson, Richard M. Logan, Gloria C. Mejia, EA Coates, AD Freeman, Liberali, SA, Coates, EA, Freeman, AD, Logan, RM, Jamieson, L, and Mejia, G
- Subjects
Adult ,Male ,medicine.medical_specialty ,Cross-sectional study ,Human immunodeficiency virus (HIV) ,MEDLINE ,Pain ,Dentistry ,Oral Health ,Dental Caries ,medicine.disease_cause ,Xerostomia ,Young Adult ,Quality of life (healthcare) ,Internal medicine ,Statistical significance ,Dentistry, Oral Surgery & Medicine ,HIV Seropositivity ,South Australia ,Prevalence ,CART ,Humans ,Medicine ,Young adult ,General Dentistry ,Periodontal Diseases ,Health Services Needs and Demand ,AIDS-Related Opportunistic Infections ,DMF Index ,business.industry ,Smoking ,Social impact ,virus diseases ,Middle Aged ,HIV infection ,Dental patients ,stomatognathic diseases ,Cross-Sectional Studies ,Anti-Retroviral Agents ,Quality of Life ,oral health ,Female ,Mouth Diseases ,business - Abstract
Background A study undertaken in 1992–1993 identified that HIV-infected dental patients were substantially disadvantaged with regard to the social impact of their oral disease. The oral pain experienced by HIV-positive patients prior to the introduction of combination antiretroviral therapy (cART) was attributable to specific features of HIV-related periodontal disease and other oral manifestations of HIV such as candida infections and xerostomia. A repeat of this study in 2009–2010 provided additional information in the post-cART era. Methods Data were collected from three sources: the 2009–2010 HIV-positive sample, the National Survey of Adult Oral Health (NSAOH) and the original 1992–1993 study. Collation of data was by clinical and radiographic oral examination. Information about the social impact of oral conditions was obtained from the Oral Health Impact Profile. Results The caries experience of the 2009–2010 HIV-positive sample was improved with statistical significance for both mean DMFT and mean DT, while the presence of HIV-related periodontal disease still occurs. Statistically significant improvements were achieved for prevalence and severity of oral health related quality of life. Conclusions The need for timely access to oral health care with a focus on prevention is essential for HIV-positive individuals whose health is impacted by chronic disease, smoking and salivary hypofunction.
- Published
- 2013
5. Adverse Tracheal Intubation Events in Critically Ill Underweight and Obese Children: Retrospective Study of the National Emergency Airway for Children Registry (2013-2020).
- Author
-
Gladen KM, Tellez D, Napolitano N, Edwards LR, Sanders RC Jr, Kojima T, Malone MP, Shults J, Krawiec C, Ambati S, McCarthy R, Branca A, Polikoff LA, Jung P, Parsons SJ, Mallory PP, Komeswaran K, Page-Goertz C, Toal MC, Bysani GK, Meyer K, Chiusolo F, Glater-Welt LB, Al-Subu A, Biagas K, Hau Lee J, Miksa M, Giuliano JS Jr, Kierys KL, Talukdar AM, DeRusso M, Cucharme-Crevier L, Adu-Arko M, Shenoi AN, Kimura D, Flottman M, Gangu S, Freeman AD, Piehl MD, Nuthall GA, Tarquinio KM, Harwayne-Gidansky I, Hasegawa T, Rescoe ES, Breuer RK, Kasagi M, Nadkarni VM, and Nishisaki A
- Subjects
- Infant, Child, Humans, Infant, Newborn, Child, Preschool, Adolescent, Retrospective Studies, Overweight etiology, Thinness complications, Thinness epidemiology, Intubation, Intratracheal adverse effects, Intubation, Intratracheal methods, Hypoxia epidemiology, Hypoxia etiology, Registries, Critical Illness, Pediatric Obesity complications, Pediatric Obesity epidemiology
- Abstract
Objectives: Extremes of patient body mass index are associated with difficult intubation and increased morbidity in adults. We aimed to determine the association between being underweight or obese with adverse airway outcomes, including adverse tracheal intubation (TI)-associated events (TIAEs) and/or severe peri-intubation hypoxemia (pulse oximetry oxygen saturation < 80%) in critically ill children., Design/setting: Retrospective cohort using the National Emergency Airway for Children registry dataset of 2013-2020., Patients: Critically ill children, 0 to 17 years old, undergoing TI in PICUs., Interventions: None., Measurements and Main Results: Registry data from 24,342 patients who underwent TI between 2013 and 2020 were analyzed. Patients were categorized using the Centers for Disease Control and Prevention weight-for-age chart: normal weight (5th-84th percentile) 57.1%, underweight (< 5th percentile) 27.5%, overweight (85th to < 95th percentile) 7.2%, and obese (≥ 95th percentile) 8.2%. Underweight was most common in infants (34%); obesity was most common in children older than 8 years old (15.1%). Underweight patients more often had oxygenation and ventilation failure (34.0%, 36.2%, respectively) as the indication for TI and a history of difficult airway (16.7%). Apneic oxygenation was used more often in overweight and obese patients (19.1%, 19.6%) than in underweight or normal weight patients (14.1%, 17.1%; p < 0.001). TIAEs and/or hypoxemia occurred more often in underweight (27.1%) and obese (24.3%) patients ( p < 0.001). TI in underweight children was associated with greater odds of adverse airway outcome compared with normal weight children after adjusting for potential confounders (underweight: adjusted odds ratio [aOR], 1.09; 95% CI, 1.01-1.18; p = 0.016). Both underweight and obesity were associated with hypoxemia after adjusting for covariates and site clustering (underweight: aOR, 1.11; 95% CI, 1.02-1.21; p = 0.01 and obesity: aOR, 1.22; 95% CI, 1.07-1.39; p = 0.002)., Conclusions: In underweight and obese children compared with normal weight children, procedures around the timing of TI are associated with greater odds of adverse airway events., Competing Interests: Ms. Napolitano’s, Dr. Shults’s, Dr. Nadkarni’s, and Dr. Nishisaki’s institution received funding from the Agency for Healthcare Research and Quality (R18HS024511). Ms. Napolitano’s institution received funding from Dräger, Actuated Medical, and Philips Respironics; they received support for article research from Timpel and VERO-Biotech. Dr. Krawiec received funding from New England Journal of Medicine Healer Cases and Carle Illinois School of Medicine Admissions Committee. Dr. Polikoff received funding from Novavax. Dr. Lee’s institution received funding from National Research Medical Council, Singapore. Dr. Shenoi is an elected member of the Society of Critical Care Medicine (SCCM) Council of the SCCM. Dr. Peihl disclosed that he is founder and Chief Medical Officer of 410 Medical; his institution received funding the Department of Defense (grants: USSOCOM W81XWH-22-C-0002, USAF SBIR AF212-CSO1 Phase II, F2-15653 USAF, SBIR AF211-CSO1 Phase II, F2-15254). Dr. Hasegawa disclosed work for hire. Dr. Nishisaki’s institution received funding from Chiesi USA, AHRA, and the National Institute of Child Health and Human Development. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2023 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)
- Published
- 2024
- Full Text
- View/download PDF
6. An inactivated multivalent influenza A virus vaccine is broadly protective in mice and ferrets.
- Author
-
Park J, Fong Legaspi SL, Schwartzman LM, Gygli SM, Sheng ZM, Freeman AD, Matthews LM, Xiao Y, Ramuta MD, Batchenkova NA, Qi L, Rosas LA, Williams SL, Scherler K, Gouzoulis M, Bellayr I, Morens DM, Walters KA, Memoli MJ, Kash JC, and Taubenberger JK
- Subjects
- Animals, Antibodies, Viral, Ferrets, Horses, Humans, Influenza A Virus, H7N3 Subtype, Mice, Swine, Influenza A Virus, H1N1 Subtype, Influenza A Virus, H3N8 Subtype, Influenza A Virus, H5N1 Subtype, Influenza A Virus, H7N9 Subtype, Influenza Vaccines, Orthomyxoviridae Infections
- Abstract
Influenza A viruses (IAVs) present major public health threats from annual seasonal epidemics and pandemics and from viruses adapted to a variety of animals including poultry, pigs, and horses. Vaccines that broadly protect against all such IAVs, so-called "universal" influenza vaccines, do not currently exist but are urgently needed. Here, we demonstrated that an inactivated, multivalent whole-virus vaccine, delivered intramuscularly or intranasally, was broadly protective against challenges with multiple IAV hemagglutinin and neuraminidase subtypes in both mice and ferrets. The vaccine is composed of four β-propiolactone-inactivated low-pathogenicity avian IAV subtypes of H1N9, H3N8, H5N1, and H7N3. Vaccinated mice and ferrets demonstrated substantial protection against a variety of IAVs, including the 1918 H1N1 strain, the highly pathogenic avian H5N8 strain, and H7N9. We also observed protection against challenge with antigenically variable and heterosubtypic avian, swine, and human viruses. Compared to control animals, vaccinated mice and ferrets demonstrated marked reductions in viral titers, lung pathology, and host inflammatory responses. This vaccine approach indicates the feasibility of eliciting broad, heterosubtypic IAV protection and identifies a promising candidate for influenza vaccine clinical development.
- Published
- 2022
- Full Text
- View/download PDF
7. Pre-existing immunity to influenza virus hemagglutinin stalk might drive selection for antibody-escape mutant viruses in a human challenge model.
- Author
-
Park JK, Xiao Y, Ramuta MD, Rosas LA, Fong S, Matthews AM, Freeman AD, Gouzoulis MA, Batchenkova NA, Yang X, Scherler K, Qi L, Reed S, Athota R, Czajkowski L, Han A, Morens DM, Walters KA, Memoli MJ, Kash JC, and Taubenberger JK
- Subjects
- Animals, Antibodies, Neutralizing genetics, Antibodies, Neutralizing immunology, Antibodies, Neutralizing pharmacology, Antibodies, Viral genetics, Antibodies, Viral immunology, Antibodies, Viral pharmacology, Conserved Sequence genetics, Cross Reactions immunology, Hemagglutinin Glycoproteins, Influenza Virus immunology, Humans, Influenza A Virus, H1N1 Subtype immunology, Influenza A Virus, H1N1 Subtype pathogenicity, Influenza Vaccines genetics, Influenza Vaccines immunology, Influenza, Human immunology, Influenza, Human prevention & control, Influenza, Human virology, Mice, Selection, Genetic immunology, Hemagglutinin Glycoproteins, Influenza Virus genetics, Influenza A Virus, H1N1 Subtype genetics, Influenza, Human genetics, Selection, Genetic genetics
- Abstract
The conserved region of influenza hemagglutinin (HA) stalk (or stem) has gained attention as a potent target for universal influenza vaccines
1-5 . Although the HA stalk region is relatively well conserved, the evolutionarily dynamic nature of influenza viruses6 raises concerns about the possible emergence of viruses carrying stalk escape mutation(s) under sufficient immune pressure. Here we show that immune pressure on the HA stalk can lead to expansion of escape mutant viruses in study participants challenged with a 2009 H1N1 pandemic influenza virus inoculum containing an A388V polymorphism in the HA stalk (45% wild type and 55% mutant). High level of stalk antibody titers was associated with the selection of the mutant virus both in humans and in vitro. Although the mutant virus showed slightly decreased replication in mice, it was not observed in cell culture, ferrets or human challenge participants. The A388V mutation conferred resistance to some of the potent HA stalk broadly neutralizing monoclonal antibodies (bNAbs). Co-culture of wild-type and mutant viruses in the presence of either a bNAb or human serum resulted in rapid expansion of the mutant. These data shed light on a potential obstacle for the success of HA-stalk-targeting universal influenza vaccines-viral escape from vaccine-induced stalk immunity.- Published
- 2020
- Full Text
- View/download PDF
8. Automated Pupillary Measurements Inversely Correlate With Increased Intracranial Pressure in Pediatric Patients With Acute Brain Injury or Encephalopathy.
- Author
-
Freeman AD, McCracken CE, and Stockwell JA
- Subjects
- Child, Humans, Intracranial Pressure, Prospective Studies, Pupil, Reflex, Pupillary, Brain Injuries complications, Brain Injuries diagnosis, Pediatrics
- Abstract
Objectives: The purpose of this study was to determine correlation and temporal association between automated pupillary measurements and intracranial pressure in pediatric patients with brain injury or encephalopathy requiring intracranial pressure monitoring. We hypothesized that abnormal pupillary measurements would precede increases in intracranial pressure., Design: A prospective cohort study was performed. Automated pupillometry measurements were obtained at the same frequency as the patients' neurologic assessments with concurrent measurement of intracranial pressure, for up to 72 hours. Pupillary measurements and the Neurologic Pupil index, an algorithmic score that combines measures of pupillary reactivity, were assessed for correlation with concurrent and future intracranial pressure measurements., Setting: Single-center pediatric quaternary ICU, from July 2017 to October 2018., Patients: Pediatric patients 18 years or younger with a diagnosis of acute brain injury or encephalopathy requiring an intracranial pressure monitor., Interventions: None., Measurements and Main Results: Twenty-eight patients were analyzed with a total of 1,171 intracranial pressure measurements. When intracranial pressure was elevated, the Neurologic Pupil index, percent change in pupillary size, constriction velocity, and dilation velocity were significantly lower than when intracranial pressure was within normal range (p < 0.001 for all). There were mild to moderate negative correlations between concurrent intracranial pressure and pupillary measurements. However, there was an inconsistent pattern of abnormal pupillary measurements preceding increases in intracranial pressure; some patients had a negative association, while others had a positive relationship or no relationship between Neurologic Pupil index and intracranial pressure., Conclusions: Our data indicate automated assessments of pupillary reactivity inversely correlate with intracranial pressure, demonstrating that pupillary reactivity decreases as intracranial pressure increases. However, a temporal association in which abnormal pupillary measurements precede increases in intracranial pressure was not consistently observed. This work contributes to limited data available regarding automated pupillometry in neurocritically ill patients, and the even more restricted subset available in pediatrics.
- Published
- 2020
- Full Text
- View/download PDF
9. Does hypertension at initial presentation adversely affect outcomes in pediatric traumatic brain injury?
- Author
-
Freeman AD, Fitzgerald CA, Baxter KJ, Neff LP, McCracken CE, Bryan LN, Morsberger JL, Zahid AM, and Santore MT
- Subjects
- Blood Pressure, Brain Injuries, Traumatic rehabilitation, Brain Injuries, Traumatic surgery, Child, Child, Preschool, Female, Humans, Infant, Length of Stay, Male, Neurological Rehabilitation, Neurosurgical Procedures statistics & numerical data, Respiration, Artificial, Retrospective Studies, Time Factors, Trauma Severity Indices, Brain Injuries, Traumatic complications, Brain Injuries, Traumatic mortality, Hypertension complications
- Abstract
Background: Adults with traumatic brain injury (TBI) who present hypertensive suffer worse outcomes and increased mortality compared to normotensive patients. The purpose of this study is to determine if age-adjusted hypertension on presentation is associated with worsened outcomes in pediatric TBI., Methods: A retrospective chart review was conducted on pediatric patients with severe TBI admitted to a single system pediatric tertiary care center. The primary outcome was mortality. Secondary outcomes included length of stay, need for neurosurgical intervention, duration of mechanical ventilation, and the need for inpatient rehabilitation., Results: Of 150 patients, 70% were hypertensive and 30% were normotensive on presentation. Comparing both groups, no statistically significant differences were noted in mortality (13.3% for both groups), need for neurosurgical intervention (51.4% vs 48.8%, p = 0.776), length of stay (6 vs 8 days, p = 0.732), duration of mechanical ventilation (2 vs 3 days, p = 0.912), or inpatient rehabilitation rates (48.6% vs 48.9%, p = 0.972). In comparing just the hypertensive patients, there was a trend toward increased mortality in the 95th and 99th percentile groups at 15.8% and 14.1%, versus the 90th percentile group at 6.7% but the difference was not statistically significant (p = 0.701)., Conclusions: Contrary to the adult literature, pediatric patients with severe TBI and hypertension on presentation do not appear to have worsened outcomes compared to those who are normotensive. However, a trend toward increased mortality did exist at extremes of age adjusted hypertension. Larger scale studies are needed to validate these findings., Study Type: Retrospective cohort study LEVEL OF EVIDENCE: III., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
10. A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1.
- Author
-
Liu Y, Freeman AD, Déclais AC, and Lilley DMJ
- Subjects
- Catalytic Domain genetics, Chaetomium genetics, Chaetomium metabolism, Cloning, Molecular, Crystallography, X-Ray, DNA Cleavage, DNA, Cruciform metabolism, Escherichia coli, Holliday Junction Resolvases genetics, Ions chemistry, Protein Binding, Substrate Specificity genetics, Chaetomium enzymology, DNA, Fungal metabolism, Holliday Junction Resolvases chemistry, Holliday Junction Resolvases metabolism, Protein Interaction Domains and Motifs genetics
- Abstract
GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.
- Published
- 2018
- Full Text
- View/download PDF
11. Analysis of the Intrinsically Disordered N-Terminus of the DNA Junction-Resolving Enzyme T7 Endonuclease I: Identification of Structure Formed upon DNA Binding.
- Author
-
Freeman AD, Stevens M, Declais AC, Leahy A, Mackay K, El Mkami H, Lilley DM, and Norman DG
- Subjects
- Bacteriophage T7 genetics, Deoxyribonuclease I genetics, Electron Spin Resonance Spectroscopy, Intrinsically Disordered Proteins genetics, Models, Molecular, Mutagenesis, Site-Directed, Nucleic Acid Conformation, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Viral Proteins genetics, Bacteriophage T7 enzymology, DNA, Cruciform chemistry, DNA, Cruciform metabolism, Deoxyribonuclease I chemistry, Deoxyribonuclease I metabolism, Intrinsically Disordered Proteins chemistry, Intrinsically Disordered Proteins metabolism, Viral Proteins chemistry, Viral Proteins metabolism
- Abstract
The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process. Here we have investigated the N-terminal peptide by inclusion of spin-label probes at unique sites within this region, studied by electron paramagnetic resonance. Continuous wave experiments show that these labels are mobile in the free protein but become constrained on binding a DNA junction, with the main interaction occurring for residues 7-10 and 12. Distance measurements between equivalent positions within the two peptides of a dimer using PELDOR showed that the intermonomeric distances for residues 2-12 are long and broadly distributed in the free protein but are significantly shortened and become more defined on binding to DNA. These results suggest that the N-terminal peptides become more organized on binding to the DNA junction and nestle into the minor grooves at the branchpoint, consistent with the biochemical data indicating an important role in the resolution process. This study demonstrates the presence of structure within a protein region that cannot be viewed by crystallography.
- Published
- 2016
- Full Text
- View/download PDF
12. The importance of the N-terminus of T7 endonuclease I in the interaction with DNA junctions.
- Author
-
Freeman AD, Déclais AC, and Lilley DM
- Subjects
- Binding Sites, DNA Primers chemistry, DNA, Bacterial chemistry, Deoxyribonuclease I chemistry, Deoxyribonuclease I genetics, Models, Molecular, Nucleic Acid Conformation, Peptide Fragments chemistry, Peptide Fragments genetics, Protein Binding, Bacteriophage T7 enzymology, DNA, Bacterial metabolism, Deoxyribonuclease I metabolism, Peptide Fragments metabolism
- Abstract
T7 endonuclease I is a dimeric nuclease that is selective for four-way DNA junctions. Previous crystallographic studies have found that the N-terminal 16 amino acids are not visible, neither in the presence nor in the absence of DNA. We have now investigated the effect of deleting the N-terminus completely or partially. N-terminal deleted enzyme binds more tightly to DNA junctions but cleaves them more slowly. While deletion of the N-terminus does not measurably affect the global structure of the complex, the presence of the peptide is required to generate a local opening at the center of the DNA junction that is observed by 2-aminopurine fluorescence. Complete deletion of the peptide leads to a cleavage rate that is 3 orders of magnitude slower and an activation enthalpy that is 3-fold higher, suggesting that the most important interaction of the peptide is with the reaction transition state. Taken together, these data point to an important role of the N-terminus in generating a central opening of the junction that is required for the cleavage reaction to proceed properly. In the absence of this, we find that a cruciform junction is no longer subject to bilateral cleavage, but instead, just one strand is cleaved. Thus, the N-terminus is required for a productive resolution of the junction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
13. Single-molecule observation of the induction of k-turn RNA structure on binding L7Ae protein.
- Author
-
Wang J, Fessl T, Schroeder KT, Ouellet J, Liu Y, Freeman AD, and Lilley DM
- Subjects
- Archaeal Proteins chemistry, Archaeoglobus fulgidus, Base Sequence, Haloarcula marismortui, Immobilized Proteins chemistry, Immobilized Proteins metabolism, Protein Binding, RNA, Archaeal genetics, Archaeal Proteins metabolism, Nucleotide Motifs, RNA, Archaeal chemistry, RNA, Archaeal metabolism
- Abstract
The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
14. Analysis of conformational changes in the DNA junction-resolving enzyme T7 endonuclease I on binding a four-way junction using EPR.
- Author
-
Freeman AD, Ward R, El Mkami H, Lilley DM, and Norman DG
- Subjects
- Bacteriophage T7 chemistry, Bacteriophage T7 genetics, Deoxyribonuclease I chemistry, Deoxyribonuclease I genetics, Electron Spin Resonance Spectroscopy, Models, Molecular, Mutation, Nucleic Acid Conformation, Protein Binding, Protein Conformation, Protein Multimerization, Bacteriophage T7 enzymology, DNA metabolism, Deoxyribonuclease I metabolism
- Abstract
The four-way (Holliday) DNA junction is the central intermediate in homologous recombination. It is ultimately resolved into two nicked-duplex species by the action of a junction-resolving enzyme. These enzymes are highly selective for the structure of branched DNA, yet as a class these proteins impose significant distortion on their target junctions. Bacteriophage T7 endonuclease I selectively binds and cleaves DNA four-way junctions. The protein is an extremely stable dimer, comprising two globular domains joined by a β-strand bridge with each active site including amino acids from both polypeptides. The crystal structure of endonuclease I has been solved both as free protein and in complex with a DNA junction, showing that the protein, as well as the junction, becomes distorted on binding. We have therefore used site-specific spin-labeling in conjunction with EPR distance measurements to analyze induced fit in the binding of endonuclease I to a DNA four-way junction. The results support the change in protein structure as it binds to the junction. In addition, we have examined the structure of wild type and catalytically inactive mutants alone and in complex with DNA. We demonstrate the presence of hitherto undefined metastable conformational states within endonuclease I, showing how these states can be influenced by DNA-junction binding or mutations within the active sites. In addition, we demonstrate a previously unobserved instability in the N-terminal α1-helix upon active site mutation. These studies reveal that structural changes in both DNA and protein occur in the action of this junction-resolving enzyme., (© 2011 American Chemical Society)
- Published
- 2011
- Full Text
- View/download PDF
15. Comparing the mutagenicity of toxaphene after aging in anoxic soils and accumulating in fish.
- Author
-
Young JC, Freeman AD, Bruce RM, Williams D, and Maruya K
- Subjects
- Animals, Biodegradation, Environmental, Camphanes pharmacokinetics, Camphanes toxicity, Insecticides isolation & purification, Insecticides pharmacokinetics, Mutagenicity Tests, Toxaphene isolation & purification, Toxaphene pharmacokinetics, Fishes metabolism, Insecticides toxicity, Mutagens toxicity, Soil analysis, Toxaphene toxicity
- Abstract
A test program was conducted to evaluate the mutagenicity of toxaphene residuals extracted from aged soils and from fish collected in creeks near a toxaphene-contaminated site. The ultimate objective was to determine if the residual toxaphene congeners were more or less mutagenic than those in technical-grade toxaphene. The study showed that the mutagenicity of the bioaccumulated toxaphene congeners in fish, expressed as colony revertants per microg of residual toxaphene, was no greater than that of technical-grade toxaphene. The mutagenic impact of the toxaphene residuals in aged soil statistically was less than that for technical-grade toxaphene. Two specific congeners, a hexachlorobornane (labeled Hx-Sd) and a heptachlorobornane (labeled Hp-Sd), were found to accumulate over time in both soil and fish extracts, but did not show increased mutagenic impacts relative to that produced by technical-grade toxaphene.
- Published
- 2009
- Full Text
- View/download PDF
16. Malpractice claims involving pediatricians.
- Author
-
Freeman JM and Freeman AD
- Subjects
- Humans, Malpractice legislation & jurisprudence, Malpractice statistics & numerical data, Medical Errors statistics & numerical data, Pediatrics statistics & numerical data, United States, Compensation and Redress, Liability, Legal economics, Malpractice economics, Pediatrics legislation & jurisprudence
- Published
- 2007
- Full Text
- View/download PDF
17. Structural recognition between a four-way DNA junction and a resolving enzyme.
- Author
-
Déclais AC, Liu J, Freeman AD, and Lilley DM
- Subjects
- Base Sequence, DNA Footprinting, DNA, Circular chemistry, DNA, Cruciform genetics, Deoxyribonuclease I chemistry, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Organophosphorus Compounds metabolism, Protein Binding, Substrate Specificity, Bacteriophage T7 enzymology, DNA, Cruciform chemistry, DNA, Cruciform metabolism, Deoxyribonuclease I metabolism, Holliday Junction Resolvases chemistry, Holliday Junction Resolvases metabolism
- Abstract
Resolving enzymes bind highly selectively to four-way DNA junctions, but the mechanism of this structural specificity is poorly understood. In this study, we have explored the role of interactions between the dimeric enzyme and the helical arms of the junction, using junctions with either shortened arms, or circular permutation of arms. We find that DNA-protein contacts in the arms containing the 5' ends of the continuous strands are very important, conferring a significant level of sequence discrimination upon both the choice of conformer and the order of strand cleavage. We have exploited these properties to obtain hydroxyl radical footprinting data on endonuclease I-junction complexes that are not complicated by the presence of alternative conformers, with results that are in good agreement with the arm permutation and shortening experiments. Substitution of phosphate groups at the center of the junction reveals the importance of electrostatic interactions at the point of strand exchange in the complex. Our data show that the form of the complex between endonuclease I and a DNA junction depends on the core of the junction and on interactions with the first six base-pairs of the arms containing the 5' ends of the continuous strands.
- Published
- 2006
- Full Text
- View/download PDF
18. Observing spontaneous branch migration of Holliday junctions one step at a time.
- Author
-
McKinney SA, Freeman AD, Lilley DM, and Ha T
- Subjects
- Base Sequence, DNA, Cruciform chemistry, Fluorescence Resonance Energy Transfer, Magnesium chemistry, Oligonucleotides chemistry, Oligonucleotides genetics, Oligonucleotides metabolism, Thermodynamics, Base Pairing, DNA, Cruciform metabolism, Nucleic Acid Conformation, Recombination, Genetic
- Abstract
Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. Conventional ensemble assays, useful for finding average migration rates over long sequences, have been unable to examine the affect of sequence and structure on the migration process. Here, we present a single-molecule spontaneous branch migration assay with single-base pair resolution in a study of individual DNA junctions that can undergo one step of migration. Junctions exhibit markedly different dynamics of exchange between stacking conformers depending on the point of strand exchange, allowing the moment at which branch migration occurs to be detected. The free energy landscape of spontaneous branch migration is found to be highly nonuniform and governed by two types of sequence-dependent barriers, with unmediated local migration being up to 10 times more rapid than the previously deduced average rate.
- Published
- 2005
- Full Text
- View/download PDF
19. Metal ion binding in the active site of the junction-resolving enzyme T7 endonuclease I in the presence and in the absence of DNA.
- Author
-
Freeman AD, Déclais AC, and Lilley DM
- Subjects
- Bacteriophage T7 enzymology, Binding Sites, Calorimetry, Calcium metabolism, DNA metabolism, Deoxyribonuclease I metabolism, Iron metabolism
- Abstract
Endonuclease I of bacteriophage T7 is a DNA junction-resolving enzyme. We have previously used crystallography to demonstrate the binding of two manganese ions into the active site that is formed by three carboxylate (Glu 20, Asp 55 and Glu 65) and a lysine residue (Lys 67). Endonuclease I is active in the presence of magnesium, manganese, iron (II) and cobalt (II) ions, weakly active in the presence of nickel, copper (II) and zinc ions, and completely inactive in the presence of calcium ions. However, using calorimetry, we have observed the binding of two calcium ions to the free enzyme in a manner very similar to the binding of manganese ions. In the presence of iron (II) ions, we have obtained a cleavage of the continuous strands of a junction bound by endonuclease I, at sites close to (but not identical with) enzyme-induced hydrolysis. The results suggest that this arises from attack by locally generated hydroxyl radicals, arising from iron (II) ions bound into the active site. This therefore provides an indirect way of examining metal ion binding in the enzyme-junction complex. Ion binding in free protein (by calorimetry) and the enzyme-junction complex (iron-induced cleavage) have been studied in series of active-site mutants. Both confirm the importance of the three carboxylate ligands, and the lack of a requirement for Lys67 for the ion binding. Calorimetry points to particularly critical role of Asp55, as mutation completely abolishes all binding of both manganese and calcium ions.
- Published
- 2003
- Full Text
- View/download PDF
20. The complex between a four-way DNA junction and T7 endonuclease I.
- Author
-
Déclais AC, Fogg JM, Freeman AD, Coste F, Hadden JM, Phillips SE, and Lilley DM
- Subjects
- 2-Aminopurine metabolism, Binding Sites, Calcium metabolism, Cations, Divalent metabolism, DNA Repair, DNA, Bacterial chemistry, DNA, Bacterial genetics, Deoxyribonuclease I chemistry, Deoxyribonuclease I genetics, Deoxyribonuclease I isolation & purification, Hydroxyl Radical metabolism, Kinetics, Magnesium metabolism, Manganese Compounds metabolism, Models, Molecular, Nucleic Acid Conformation, Oxides metabolism, Phosphorus Radioisotopes, Protein Binding, Recombination, Genetic, Thymine metabolism, Bacteriophage T7 enzymology, DNA, Bacterial metabolism, Deoxyribonuclease I metabolism
- Abstract
The junction-resolving enzyme endonuclease I is selective for the structure of the DNA four-way (Holliday) junction. The enzyme binds to a four-way junction in two possible orientations, with a 4:1 ratio, opening the DNA structure at the centre and changing the global structure into a 90 degrees cross of approximately coaxial helices. The nuclease cleaves the continuous strands of the junction in each orientation. Binding leads to pronounced regions of protection of the DNA against hydroxyl radical attack. Using all this information together with the known structure of the enzyme and the structure of the BglI-DNA complex, we have constructed a model of the complex of endonuclease I and a DNA junction. This shows how the enzyme is selective for the structure of a four-way junction, such that both continuous strands can be accommodated into the two active sites so that a productive resolution event is possible.
- Published
- 2003
- Full Text
- View/download PDF
21. Cerebral palsy and the 'bad baby' malpractice crisis. New York State shines light toward the end of the tunnel.
- Author
-
Freeman JM and Freeman AD
- Subjects
- Asphyxia Neonatorum blood, Asphyxia Neonatorum diagnosis, Cerebral Palsy epidemiology, Cerebral Palsy prevention & control, Expert Testimony legislation & jurisprudence, Fetal Blood chemistry, Fetal Monitoring methods, Fetal Monitoring standards, Humans, Hydrogen-Ion Concentration, Infant, Newborn, Liability, Legal, Malpractice legislation & jurisprudence, Malpractice trends, New York epidemiology, Obstetrics legislation & jurisprudence, Obstetrics standards, Obstetrics statistics & numerical data, Pediatrics legislation & jurisprudence, Pediatrics standards, Pediatrics statistics & numerical data, Physician's Role, Asphyxia Neonatorum complications, Cerebral Palsy etiology, Malpractice statistics & numerical data
- Published
- 1992
- Full Text
- View/download PDF
22. Ontogenetic development of histamine H1-receptor binding in rat brain.
- Author
-
Tran VT, Freeman AD, Chang RS, and Snyder SH
- Subjects
- Aging, Animals, Binding, Competitive, Brain metabolism, Histamine H1 Antagonists pharmacology, Kinetics, Protein Binding, Pyrilamine metabolism, Rats, Brain growth & development, Receptors, Histamine metabolism, Receptors, Histamine H1 metabolism
- Published
- 1980
- Full Text
- View/download PDF
23. No-fault cerebral palsy insurance: an alternative to the obstetrical malpractice lottery.
- Author
-
Freeman AD and Freeman JM
- Subjects
- Female, Humans, Infant, Newborn, Pregnancy, United States, Birth Injuries economics, Cerebral Palsy economics, Insurance, Liability organization & administration, Malpractice economics, Obstetrics economics
- Abstract
Sixty percent of malpractice premiums paid by obstetricians go to cover suits for alleged birth-related cerebral palsy (CP). Yet substantially less than half of that money goes to CP victims, and less than 10 percent of children with CP receive any compensation at all from tort suits. This paper proposes a system that would compensate all children born with CP for most handicap-related expenses, in exchange for which the children would be foreclosed from bringing suits alleging birth-related malpractice. Malpractice would be policed by a state board, which would investigate all CP cases. This proposal would be more equitable than current systems. It would also be less expensive, since it would avoid costly litigation and decrease the cost of obstetrical malpractice insurance.
- Published
- 1989
- Full Text
- View/download PDF
24. People types and productivity.
- Author
-
Freeman AD
- Subjects
- Humans, Personality Inventory, Personnel Administration, Hospital, Efficiency, Personality, Personnel Management
- Published
- 1988
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.