mRNA quantification and clinical evaluation of telomerase reverse transcriptase subunit (hTERT) in intracranial tumours of patients in the island of Crete

Telomerase is a reverse transcriptase that maintains telomeres by adding telomeric TTAGGG repeats to the ends of human chromosomes. The aim of this study was to evaluate quantitatively the mRNA expression of telomerase catalytic subunit (hTERT) in different types of intracranial tumours in relation to their histologic pattern and grade and correlate it with progression-free (PFS) and overall survival (OS) of patients. Human telomerase reverse transcriptase mRNA levels were estimated by the use of real time RT–PCR in 68 samples of intracranial tumours. It revealed statistical correlation between hTERT mRNA expression levels and the grade of the tumours (P<0.001). Patients having negative expression of hTERT mRNA had statistically longer PFS (P=0.031) and OS (P=0.047). Cox univariate regression analysis revealed that hTERT mRNA-positive patients had a high and statistically significant risk of relapse (hazard ratio (HR) of 2.24 and P=0.038). In the Cox multivariate regression model, the levels of hTERT mRNA were adjusted for tumour grade and patients age, and since there was statistically significant relationship between the levels of hTERT mRNA and the grade of the tumours (P=0.003 or P=0.006, respectively), hTERT mRNA levels could not be considered as an independent prognostic factor for PFS or OS

Troglitazone suppresses telomerase activity independently of PPARγ in estrogen-receptor negative breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Breast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARγ). However, its effect on telomerase regulation in breast cancer has not been investigated.</p> <p>Methods</p> <p>In this study, we investigated the effect of the PPARγ ligand, troglitazone, on telomerase activity in the MDA-MB-231 breast cancer cell line. Real time RT-PCR and telomerase activity assays were used to evaluate the effect of troglitazone. MDA-MB-231 cells had PPARγ expression silenced using shRNA interference.</p> <p>Results</p> <p>We demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Troglitazone reduced telomerase activity even in the absence of PPARγ. In agreement with this result, we found no correlation between PPARγ and hTERT mRNA transcript levels in breast cancer patients. Statistical significance was determined using Pearson correlation and the paired Student's <it>t </it>test.</p> <p>Conclusions</p> <p>To our knowledge, this is the first time that the effect of troglitazone on telomerase activity in breast cancer cells has been investigated. Our data suggest that troglitazone may be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined.</p

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

The Secreted Lipoprotein, MPT83, of Mycobacterium tuberculosis Is Recognized during Human Tuberculosis and Stimulates Protective Immunity in Mice

The long-term control of tuberculosis (TB) will require the development of more effective anti-TB vaccines, as the only licensed vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), has limited protective efficacy against infectious pulmonary TB. Subunit vaccines have an improved safety profile over live, attenuated vaccines, such as BCG, and may be used in immuno-compromised individuals. MPT83 (Rv2873) is a secreted mycobacterial lipoprotein expressed on the surface of Mycobacterium tuberculosis. In this study, we examined whether recombinant MPT83 is recognized during human and murine M. tuberculosis infection. We assessed the immunogenicity and protective efficacy of MPT83 as a protein vaccine, with monophosphyl lipid A (MPLA) in dimethyl-dioctadecyl ammonium bromide (DDA) as adjuvant, or as a DNA vaccine in C57BL/6 mice and mapped the T cell epitopes with peptide scanning. We demonstrated that rMPT83 was recognised by strong proliferative and Interferon (IFN)-γ-secreting T cell responses in peripheral blood mononuclear cells (PBMC) from patients with active TB, but not from healthy, tuberculin skin test-negative control subjects. MPT83 also stimulated strong IFN-γ T cell responses during experimental murine M. tuberculosis infection. Immunization with either rMPT83 in MPLA/DDA or DNA-MPT83 stimulated antigen-specific T cell responses, and we identified MPT83127–135 (PTNAAFDKL) as the dominant H-2b-restricted CD8+ T cell epitope within MPT83. Further, immunization of C57BL/6 mice with rMPT83/MPLA/DDA or DNA-MPT83 stimulated significant levels of protection in the lungs and spleens against aerosol challenge with M. tuberculosis. Interestingly, immunization with rMPT83 in MPLA/DDA primed for stronger IFN-γ T cell responses to the whole protein following challenge, while DNA-MPT83 primed for stronger CD8+ T cell responses to MPT83127–135. Therefore MPT83 is a protective T cell antigen commonly recognized during human M. tuberculosis infection and should be considered for inclusion in future TB subunit vaccines

Promoting advance planning for health care and research among older adults: A randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Family members are often required to act as substitute decision-makers when health care or research participation decisions must be made for an incapacitated relative. Yet most families are unable to accurately predict older adult preferences regarding future health care and willingness to engage in research studies. Discussion and documentation of preferences could improve proxies' abilities to decide for their loved ones. This trial assesses the efficacy of an advance planning intervention in improving the accuracy of substitute decision-making and increasing the frequency of documented preferences for health care and research. It also investigates the financial impact on the healthcare system of improving substitute decision-making.</p> <p>Methods/Design</p> <p>Dyads (<it>n </it>= 240) comprising an older adult and his/her self-selected proxy are randomly allocated to the experimental or control group, after stratification for type of designated proxy and self-report of prior documentation of healthcare preferences. At baseline, clinical and research vignettes are used to elicit older adult preferences and assess the ability of their proxy to predict those preferences. Responses are elicited under four health states, ranging from the subject's current health state to severe dementia. For each state, we estimated the public costs of the healthcare services that would typically be provided to a patient under these scenarios. Experimental dyads are visited at home, twice, by a specially trained facilitator who communicates the dyad-specific results of the concordance assessment, helps older adults convey their wishes to their proxies, and offers assistance in completing a guide entitled <it>My Preferences </it>that we designed specifically for that purpose. In between these meetings, experimental dyads attend a group information session about <it>My Preferences</it>. Control dyads attend three monthly workshops aimed at promoting healthy behaviors. Concordance assessments are repeated at the end of the intervention and 6 months later to assess improvement in predictive accuracy and cost savings, if any. Copies of completed guides are made at the time of these assessments.</p> <p>Discussion</p> <p>This study will determine whether the tested intervention guides proxies in making decisions that concur with those of older adults, motivates the latter to record their wishes in writing, and yields savings for the healthcare system.</p> <p>Trial Registration</p> <p><a href="http://www.controlled-trials.com/ISRCTN89993391">ISRCTN89993391</a></p

Slime mould: The fundamental mechanisms of biological cognition

© 2018 Elsevier B.V. The slime mould Physarum polycephalum has been used in developing unconventional computing devices for in which the slime mould played a role of a sensing, actuating, and computing device. These devices treated the slime mould as an active living substrate, yet it is a self-consistent living creature which evolved over millions of years and occupied most parts of the world, but in any case, that living entity did not own true cognition, just automated biochemical mechanisms. To “rehabilitate” slime mould from the rank of a purely living electronics element to a “creature of thoughts” we are analyzing the cognitive potential of P. polycephalum. We base our theory of minimal cognition of the slime mould on a bottom-up approach, from the biological and biophysical nature of the slime mould and its regulatory systems using frameworks such as Lyon's biogenic cognition, Muller, di Primio-Lengelerś modifiable pathways, Bateson's “patterns that connect” framework, Maturana's autopoietic network, or proto-consciousness and Morgan's Canon

Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer

Background Limitless self-renewal is one of the hallmarks of cancer and is attained by telomere maintenance, essentially through telomerase (hTERT) activation. Transcriptional regulation of hTERT is believed to play a major role in telomerase activation in human cancers. Main body The dominant interest in telomerase results from its role in cancer. The role of telomeres and telomere maintenance mechanisms is well established as a major driving force in generating chromosomal and genomic instability. Cancer cells have acquired the ability to overcome their fate of senescence via telomere length maintenance mechanisms, mainly by telomerase activation. hTERT expression is up-regulated in tumors via multiple genetic and epigenetic mechanisms including hTERT amplifications, hTERT structural variants, hTERT promoter mutations and epigenetic modifications through hTERT promoter methylation. Genetic (hTERT promoter mutations) and epigenetic (hTERT promoter methylation and miRNAs) events were shown to have clinical implications in cancers that depend on hTERT activation. Knowing that telomeres are crucial for cellular self-renewal, the mechanisms responsible for telomere maintenance have a crucial role in cancer diseases and might be important oncological biomarkers. Thus, rather than quantifying TERT expression and its correlation with telomerase activation, the discovery and the assessment of the mechanisms responsible for TERT upregulation offers important information that may be used for diagnosis, prognosis, and treatment monitoring in oncology. Furthermore, a better understanding of these mechanisms may promote their translation into effective targeted cancer therapies. Conclusion Herein, we reviewed the underlying mechanisms of hTERT regulation, their role in oncogenesis, and the potential clinical applications in telomerase-dependent cancers.info:eu-repo/semantics/publishedVersio

The management of acute venous thromboembolism in clinical practice. Results from the European PREFER in VTE Registry

Venous thromboembolism (VTE) is a significant cause of morbidity and mortality in Europe. Data from real-world registries are necessary, as clinical trials do not represent the full spectrum of VTE patients seen in clinical practice. We aimed to document the epidemiology, management and outcomes of VTE using data from a large, observational database. PREFER in VTE was an international, non-interventional disease registry conducted between January 2013 and July 2015 in primary and secondary care across seven European countries. Consecutive patients with acute VTE were documented and followed up over 12 months. PREFER in VTE included 3,455 patients with a mean age of 60.8 ± 17.0 years. Overall, 53.0 % were male. The majority of patients were assessed in the hospital setting as inpatients or outpatients (78.5 %). The diagnosis was deep-vein thrombosis (DVT) in 59.5 % and pulmonary embolism (PE) in 40.5 %. The most common comorbidities were the various types of cardiovascular disease (excluding hypertension; 45.5 %), hypertension (42.3 %) and dyslipidaemia (21.1 %). Following the index VTE, a large proportion of patients received initial therapy with heparin (73.2 %), almost half received a vitamin K antagonist (48.7 %) and nearly a quarter received a DOAC (24.5 %). Almost a quarter of all presentations were for recurrent VTE, with &gt;80 % of previous episodes having occurred more than 12 months prior to baseline. In conclusion, PREFER in VTE has provided contemporary insights into VTE patients and their real-world management, including their baseline characteristics, risk factors, disease history, symptoms and signs, initial therapy and outcomes

Comparative effectiveness and safety of non-vitamin K antagonists for atrial fibrillation in clinical practice: GLORIA-AF Registry

Background and purpose: Prospectively collected data comparing the safety and effectiveness of individual non-vitamin K antagonists (NOACs) are lacking. Our objective was to directly compare the effectiveness and safety of NOACs in patients with newly diagnosed atrial fibrillation (AF). Methods: In GLORIA-AF, a large, prospective, global registry program, consecutive patients with newly diagnosed AF were followed for 3&nbsp;years. The comparative analyses for (1) dabigatran vs rivaroxaban or apixaban and (2) rivaroxaban vs apixaban were performed on propensity score (PS)-matched patient sets. Proportional hazards regression was used to estimate hazard ratios (HRs) for outcomes of interest. Results: The GLORIA-AF Phase III registry enrolled 21,300 patients between January 2014 and December 2016. Of these, 3839 were prescribed dabigatran, 4015 rivaroxaban and 4505 apixaban, with median ages of 71.0, 71.0, and 73.0&nbsp;years, respectively. In the PS-matched set, the adjusted HRs and 95% confidence intervals (CIs) for dabigatran vs rivaroxaban were, for stroke: 1.27 (0.79–2.03), major bleeding 0.59 (0.40–0.88), myocardial infarction 0.68 (0.40–1.16), and all-cause death 0.86 (0.67–1.10). For the comparison of dabigatran vs apixaban, in the PS-matched set, the adjusted HRs were, for stroke 1.16 (0.76–1.78), myocardial infarction 0.84 (0.48–1.46), major bleeding 0.98 (0.63–1.52) and all-cause death 1.01 (0.79–1.29). For the comparison of rivaroxaban vs apixaban, in the PS-matched set, the adjusted HRs were, for stroke 0.78 (0.52–1.19), myocardial infarction 0.96 (0.63–1.45), major bleeding 1.54 (1.14–2.08), and all-cause death 0.97 (0.80–1.19). Conclusions: Patients treated with dabigatran had a 41% lower risk of major bleeding compared with rivaroxaban, but similar risks of stroke, MI, and death. Relative to apixaban, patients treated with dabigatran had similar risks of stroke, major bleeding, MI, and death. Rivaroxaban relative to apixaban had increased risk for major bleeding, but similar risks for stroke, MI, and death. Registration: URL: https://www.clinicaltrials.gov. Unique identifiers: NCT01468701, NCT01671007. Date of registration: September 2013