Human Telomerase Reverse Transcriptase (hTERT) Q169 Is Essential for Telomerase Function In Vitro and In Vivo

BACKGROUND:Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS:To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE:We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Compliance with barrier precautions during paediatric trauma resuscitations

Purpose: Barrier precautions protect patients and providers from blood-borne pathogens. Although barrier precaution compliance has been shown to be low among adult trauma teams, it has not been evaluated during paediatric resuscitations in which perceived risk of disease transmission may be low. The purpose of this study was to identify factors associated with compliance with barrier precautions during paediatric trauma resuscitations. Methods: Video recordings of resuscitations performed on injured children (\u3c18 years old) were reviewed to determine compliance with an established policy requiring gowns and gloves. Depending on activation level, trauma team members included up to six physicians, four nurses, and a respiratory therapist. Multivariate logistic regression was used to determine the effect of team role, resuscitation factors, and injury mechanism on barrier precaution compliance. Results: Over twelve weeks, 1138 trauma team members participated in 128 resuscitations (4.7% penetrating injuries, 9.4% highest level activations). Compliance with barrier precautions was 81.3%, with higher compliance seen among roles primarily at the bedside compared to positions not primarily at the bedside (90.7% vs 65.1%, p\u3c0.001). Bedside residents (98.4%) and surgical fellows (97.6%) had the highest compliance, while surgical attendings (20.8%) had the lowest (p\u3c0.001). Controlling for role, increased compliance was observed during resuscitations of patients with penetrating injuries (OR=3.97 [95% CI: 1.35-11.70], p=0.01), during resuscitations triaged to the highest activation level (OR=2.61 [95% CI: 1.34-5.10], p=0.005), and among team members present before patient arrival (OR=4.14 [95% CI: 2.29-7.39], p\u3c0.001). Conclusions: Compliance with barrier precautions varies by trauma team role. Team members have higher compliance when treating children with penetrating and high acuity injuries and when arriving before the patient. Interventions integrating barrier precautions into the workflow of team members are needed to reduce this variability and improve compliance with universal precautions during paediatric trauma resuscitations. © 2012 Elsevier Ireland Ltd

Guidelines for alcohol screening in adolescent trauma patients: A report from the Pediatric Trauma Society Guidelines Committee

BACKGROUND: Alcohol misuse is an important source of preventable injuries in the adolescent population. While alcohol screening and brief interventions are required at American College of SurgeonsYaccredited trauma centers, there is no standard screening method. To develop guidelines for testing, we reviewed available evidence regarding adolescent alcohol screening after injury, focusing on the questions of which populations require screening, which screening tools are most effective, and at which time point screening should be performed. METHODS: A comprehensive PubMed search for articles related to alcoholism, trauma, and screening resulted in 1,013 article abstracts for review. Eighty-five full-length articles were considered for inclusion. Articles were excluded based on study type, location (non-US), year of publication, and nonapplicability to the study questions. RESULTS: Twenty-six articles met full inclusion criteria. Results support universal screening for alcohol misuse in the adolescent trauma population. Although adolescents 14 years and older are more likely to test positive for alcohol misuse, studies suggest screening may need to start at 12 years or younger. Both survey and biochemical screens can identify at-risk adolescents, with the Alcohol Use Disorders Identification Test and the two-question survey based on the Diagnostic and Statistical Manual for Mental Disorders, Fourth Edition criteria for alcohol-use disorders being the most sensitive surveys available. CONCLUSION: Injured adolescent trauma patients should be universally screened for alcohol misuse during their hospital visit. To maximize the number of at-risk adolescents targeted for interventions, screening should begin at minimum at 12 years. As no screen identifies all atrisk adolescents, a serial screening method using both biochemical tests and standardized questionnaires may increase screening efficacy. Copyright © 2013 Lippincott Williams &Wilkins

Compliance with barrier precautions during paediatric trauma resuscitations

Purpose: Barrier precautions protect patients and providers from blood-borne pathogens. Although barrier precaution compliance has been shown to be low among adult trauma teams, it has not been evaluated during paediatric resuscitations in which perceived risk of disease transmission may be low. The purpose of this study was to identify factors associated with compliance with barrier precautions during paediatric trauma resuscitations. Methods: Video recordings of resuscitations performed on injured children (\u3c18 years old) were reviewed to determine compliance with an established policy requiring gowns and gloves. Depending on activation level, trauma team members included up to six physicians, four nurses, and a respiratory therapist. Multivariate logistic regression was used to determine the effect of team role, resuscitation factors, and injury mechanism on barrier precaution compliance. Results: Over twelve weeks, 1138 trauma team members participated in 128 resuscitations (4.7% penetrating injuries, 9.4% highest level activations). Compliance with barrier precautions was 81.3%, with higher compliance seen among roles primarily at the bedside compared to positions not primarily at the bedside (90.7% vs 65.1%, p\u3c0.001). Bedside residents (98.4%) and surgical fellows (97.6%) had the highest compliance, while surgical attendings (20.8%) had the lowest (p\u3c0.001). Controlling for role, increased compliance was observed during resuscitations of patients with penetrating injuries (OR=3.97 [95% CI: 1.35-11.70], p=0.01), during resuscitations triaged to the highest activation level (OR=2.61 [95% CI: 1.34-5.10], p=0.005), and among team members present before patient arrival (OR=4.14 [95% CI: 2.29-7.39], p\u3c0.001). Conclusions: Compliance with barrier precautions varies by trauma team role. Team members have higher compliance when treating children with penetrating and high acuity injuries and when arriving before the patient. Interventions integrating barrier precautions into the workflow of team members are needed to reduce this variability and improve compliance with universal precautions during paediatric trauma resuscitations. © 2012 Elsevier Ireland Ltd

Guidelines for alcohol screening in adolescent trauma patients: A report from the Pediatric Trauma Society Guidelines Committee

BACKGROUND: Alcohol misuse is an important source of preventable injuries in the adolescent population. While alcohol screening and brief interventions are required at American College of SurgeonsYaccredited trauma centers, there is no standard screening method. To develop guidelines for testing, we reviewed available evidence regarding adolescent alcohol screening after injury, focusing on the questions of which populations require screening, which screening tools are most effective, and at which time point screening should be performed. METHODS: A comprehensive PubMed search for articles related to alcoholism, trauma, and screening resulted in 1,013 article abstracts for review. Eighty-five full-length articles were considered for inclusion. Articles were excluded based on study type, location (non-US), year of publication, and nonapplicability to the study questions. RESULTS: Twenty-six articles met full inclusion criteria. Results support universal screening for alcohol misuse in the adolescent trauma population. Although adolescents 14 years and older are more likely to test positive for alcohol misuse, studies suggest screening may need to start at 12 years or younger. Both survey and biochemical screens can identify at-risk adolescents, with the Alcohol Use Disorders Identification Test and the two-question survey based on the Diagnostic and Statistical Manual for Mental Disorders, Fourth Edition criteria for alcohol-use disorders being the most sensitive surveys available. CONCLUSION: Injured adolescent trauma patients should be universally screened for alcohol misuse during their hospital visit. To maximize the number of at-risk adolescents targeted for interventions, screening should begin at minimum at 12 years. As no screen identifies all atrisk adolescents, a serial screening method using both biochemical tests and standardized questionnaires may increase screening efficacy. Copyright © 2013 Lippincott Williams &Wilkins

Compliance with barrier precautions during paediatric trauma resuscitations

Purpose: Barrier precautions protect patients and providers from blood-borne pathogens. Although barrier precaution compliance has been shown to be low among adult trauma teams, it has not been evaluated during paediatric resuscitations in which perceived risk of disease transmission may be low. The purpose of this study was to identify factors associated with compliance with barrier precautions during paediatric trauma resuscitations. Methods: Video recordings of resuscitations performed on injured children (\u3c18 years old) were reviewed to determine compliance with an established policy requiring gowns and gloves. Depending on activation level, trauma team members included up to six physicians, four nurses, and a respiratory therapist. Multivariate logistic regression was used to determine the effect of team role, resuscitation factors, and injury mechanism on barrier precaution compliance. Results: Over twelve weeks, 1138 trauma team members participated in 128 resuscitations (4.7% penetrating injuries, 9.4% highest level activations). Compliance with barrier precautions was 81.3%, with higher compliance seen among roles primarily at the bedside compared to positions not primarily at the bedside (90.7% vs 65.1%, p\u3c0.001). Bedside residents (98.4%) and surgical fellows (97.6%) had the highest compliance, while surgical attendings (20.8%) had the lowest (p\u3c0.001). Controlling for role, increased compliance was observed during resuscitations of patients with penetrating injuries (OR=3.97 [95% CI: 1.35-11.70], p=0.01), during resuscitations triaged to the highest activation level (OR=2.61 [95% CI: 1.34-5.10], p=0.005), and among team members present before patient arrival (OR=4.14 [95% CI: 2.29-7.39], p\u3c0.001). Conclusions: Compliance with barrier precautions varies by trauma team role. Team members have higher compliance when treating children with penetrating and high acuity injuries and when arriving before the patient. Interventions integrating barrier precautions into the workflow of team members are needed to reduce this variability and improve compliance with universal precautions during paediatric trauma resuscitations. © 2012 Elsevier Ireland Ltd