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

Insomnia symptoms and repressive coping in a sample of older Black and White women

BACKGROUND: This study examined whether ethnic differences in insomnia symptoms are mediated by differences in repressive coping styles. METHODS: A total of 1274 women (average age = 59.36 ± 6.53 years) participated in the study; 28% were White and 72% were Black. Older women in Brooklyn, NY were recruited using a stratified, cluster-sampling technique. Trained staff conducted face-to-face interviews lasting 1.5 hours acquiring sociodemographic data, health characteristics, and risk factors. A sleep questionnaire was administered and individual repressive coping styles were assessed. Fisher's exact test and Spearman and Pearson analyses were used to analyze the data. RESULTS: The rate of insomnia symptoms was greater among White women [74% vs. 46%; χ(2 )= 87.67, p < 0.0001]. Black women scored higher on the repressive coping scale than did White women [Black = 37.52 ± 6.99, White = 29.78 ± 7.38, F(1,1272 )= 304.75, p < 0.0001]. We observed stronger correlations between repressive coping and insomnia symptoms for Black [r(s )= -0.43, p < 0.0001] than for White women [r(s )= -0.18, p < 0.0001]. Controlling for variation in repressive coping, the magnitude of the correlation between ethnicity and insomnia symptoms was substantially reduced. Multivariate adjustment for differences in sociodemographics, health risk factors, physical health, and health beliefs and attitudes had little effect on the relationships. CONCLUSION: Relationships between ethnicity and insomnia symptoms are jointly dependent on the degree of repressive coping, suggesting that Black women may be reporting fewer insomnia symptoms because of a greater ability to route negative emotions from consciousness. It may be that Blacks cope with sleep problems within a positive self-regulatory framework, which allows them to deal more effectively with sleep-interfering psychological processes to stressful life events and to curtail dysfunctional sleep-interpreting processes

Quantitative Epistasis Analysis and Pathway Inference from Genetic Interaction Data

Inferring regulatory and metabolic network models from quantitative genetic interaction data remains a major challenge in systems biology. Here, we present a novel quantitative model for interpreting epistasis within pathways responding to an external signal. The model provides the basis of an experimental method to determine the architecture of such pathways, and establishes a new set of rules to infer the order of genes within them. The method also allows the extraction of quantitative parameters enabling a new level of information to be added to genetic network models. It is applicable to any system where the impact of combinatorial loss-of-function mutations can be quantified with sufficient accuracy. We test the method by conducting a systematic analysis of a thoroughly characterized eukaryotic gene network, the galactose utilization pathway in Saccharomyces cerevisiae. For this purpose, we quantify the effects of single and double gene deletions on two phenotypic traits, fitness and reporter gene expression. We show that applying our method to fitness traits reveals the order of metabolic enzymes and the effects of accumulating metabolic intermediates. Conversely, the analysis of expression traits reveals the order of transcriptional regulatory genes, secondary regulatory signals and their relative strength. Strikingly, when the analyses of the two traits are combined, the method correctly infers ∼80% of the known relationships without any false positives

United States cool surfaces deployment plan

Solar-reflective building envelope surfaces, such as cool roofs and walls, can be especially helpful in disadvantaged communities that often have poorly insulated older homes, aging or absent air conditioning units, steep utility bills, polluted air, and high vulnerability and exposure to extreme heat. With support from the U.S. Department of Energy, our project seeks to dramatically increase the climate-appropriate deployment of cool surfaces across the United States with an emphasis on their application to disadvantaged communities. First, we sought to identify cool-surface deployment barriers, opportunities, and models by (a) reviewing the history of cool-surface deployment activities, (b) interviewing cool-surface stakeholders, (c) researching successful energy-efficiency/green building deployment models; and (d) interviewing the actors who have implemented these models. Second, we conducted a workshop to engage stakeholders in development of a deployment plan. Third, we asked several U.S. federal agencies (a) how cool surfaces and cool surface stakeholders could support their missions and (b) how agency activities could support cool-surface deployment. Fourth, we identified a set of transformative ideas that form the core of the deployment plan. Transformative ideas include but are not limited to initiatives to (a) launch an educational campaign to make the general public and building professionals aware of how cool roofs and walls exclude unwanted solar heat; (b) create a “Cool Roof Prize” stimulating the development of affordable, high-performance cool asphalt roofing shingles; (c) conduct high-profile, large-scale demonstration programs that bring cool surfaces to disadvantaged heat-vulnerable communities; and (d) support local, regional, and state climate action (heat mitigation) plans with cool surfaces

Perspectives on Virtual Care for Childhood Cancer Survivors in Non-Metropolitan Areas during the COVID-19 Pandemic

The COVID-19 pandemic paved the way for the widespread use of virtual care for childhood cancer survivors (CCSs). CCSs were virtual recipients of diverse care, including long-term follow-up (LTFU), primary care, mental health care, and several others. Virtual care comes with well-documented benefits and challenges. These are further magnified for CCSs living in rural or non-metropolitan areas. Here, we describe the virtual care of CCSs from two Upper Midwest cities with well-established childhood cancer survivor programs within large comprehensive cancer centers in the United States. CCSs from non-metropolitan areas, especially CCSs with two or more late effects, used virtual care more often during the COVID-19 pandemic compared to CCSs from metropolitan areas. A review of the related literature is also included and the identified challenges in providing virtual care, such as privacy concerns, technology-connectivity constraints, and medical license restrictions. Despite these limitations, the care of CCSs has evolved to leverage virtual care and its ability to increase access for patients and promote continuity of care for CCSs living in rural areas