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

Less invasive methods of advanced hemodynamic monitoring: principles, devices, and their role in the perioperative hemodynamic optimization.

The monitoring of the cardiac output (CO) and other hemodynamic parameters, traditionally performed with the thermodilution method via a pulmonary artery catheter (PAC), is now increasingly done with the aid of less invasive and much easier to use devices. When used within the context of a hemodynamic optimization protocol, they can positively influence the outcome in both surgical and non-surgical patient populations. While these monitoring tools have simplified the hemodynamic calculations, they are subject to limitations and can lead to erroneous results if not used properly. In this article we will review the commercially available minimally invasive CO monitoring devices, explore their technical characteristics and describe the limitations that should be taken into consideration when clinical decisions are made

Potential role of differential medication use in explaining excess risk of cardiovascular events and death associated with chronic kidney disease: A cohort study

<p>Abstract</p> <p>Background</p> <p>Patients with chronic kidney disease (CKD) are less likely to receive cardiovascular medications. It is unclear whether differential cardiovascular drug use explains, in part, the excess risk of cardiovascular events and death in patients with CKD and coronary heart disease (CHD).</p> <p>Methods</p> <p>The ADVANCE Study enrolled patients with new onset CHD (2001-2003) who did (N = 159) or did not have (N = 1088) CKD at entry. The MDRD equation was used to estimate glomerular filtration rate (eGFR) using calibrated serum creatinine measurements. Patient characteristics, medication use, cardiovascular events and death were ascertained from self-report and health plan electronic databases through December 2008.</p> <p>Results</p> <p>Post-CHD event ACE inhibitor use was lower (medication possession ratio 0.50 vs. 0.58, P = 0.03) and calcium channel blocker use higher (0.47 vs. 0.38, P = 0.06) in CKD vs. non-CKD patients, respectively. Incidence of cardiovascular events and death was higher in CKD vs. non-CKD patients (13.9 vs. 11.5 per 100 person-years, P < 0.001, respectively). After adjustment for patient characteristics, the rate of cardiovascular events and death was increased for eGFR 45-59 ml/min/1.73 m²(hazard ratio [HR] 1.47, 95% CI: 1.10 to 2.02) and eGFR < 45 ml/min/1.73 m²(HR 1.58, 95% CI: 1.00 to 2.50). After further adjustment for statins, β-blocker, calcium channel blocker, ACE inhibitor/ARB use, the association was no longer significant for eGFR 45-59 ml/min/1.73 m²(HR 0.82, 95% CI: 0.25 to 2.66) or for eGFR < 45 ml/min/1.73 m²(HR 1.19, 95% CI: 0.25 to 5.58).</p> <p>Conclusions</p> <p>In adults with CHD, differential use of cardiovascular medications may contribute to the higher risk of cardiovascular events and death in patients with CKD.</p

Search for pair-produced long-lived neutral particles decaying to jets in the ATLAS hadronic calorimeter in ppcollisions at √s=8TeV

The ATLAS detector at the Large Hadron Collider at CERN is used to search for the decay of a scalar boson to a pair of long-lived particles, neutral under the Standard Model gauge group, in 20.3fb−1of data collected in proton–proton collisions at √s=8TeV. This search is sensitive to long-lived particles that decay to Standard Model particles producing jets at the outer edge of the ATLAS electromagnetic calorimeter or inside the hadronic calorimeter. No significant excess of events is observed. Limits are reported on the product of the scalar boson production cross section times branching ratio into long-lived neutral particles as a function of the proper lifetime of the particles. Limits are reported for boson masses from 100 GeVto 900 GeV, and a long-lived neutral particle mass from 10 GeVto 150 GeV

Glial Tumor Necrosis Factor Alpha (TNFα) Generates Metaplastic Inhibition of Spinal Learning

Injury-induced overexpression of tumor necrosis factor alpha (TNFα) in the spinal cord can induce chronic neuroinflammation and excitotoxicity that ultimately undermines functional recovery. Here we investigate how TNFα might also act to upset spinal function by modulating spinal plasticity. Using a model of instrumental learning in the injured spinal cord, we have previously shown that peripheral intermittent stimulation can produce a plastic change in spinal plasticity (metaplasticity), resulting in the prolonged inhibition of spinal learning. We hypothesized that spinal metaplasticity may be mediated by TNFα. We found that intermittent stimulation increased protein levels in the spinal cord. Using intrathecal pharmacological manipulations, we showed TNFα to be both necessary and sufficient for the long-term inhibition of a spinal instrumental learning task. These effects were found to be dependent on glial production of TNFα and involved downstream alterations in calcium-permeable AMPA receptors. These findings suggest a crucial role for glial TNFα in undermining spinal learning, and demonstrate the therapeutic potential of inhibiting TNFα activity to rescue and restore adaptive spinal plasticity to the injured spinal cord. TNFα modulation represents a novel therapeutic target for improving rehabilitation after spinal cord injury

Preoperative Red Cell Distribution Width and 30-day mortality in older patients undergoing non-cardiac surgery: a retrospective cohort observational study

Increased red cell distribution width (RDW) is associated with poorer outcomes in various patient populations. We investigated the association between preoperative RDW and anaemia on 30-day postoperative mortality among elderly patients undergoing non-cardiac surgery. Medical records of 24,579 patients aged 65 and older who underwent surgery under anaesthesia between 1 January 2012 and 31 October 2016 were retrospectively analysed. Patients who died within 30 days had higher median RDW (15.0%) than those who were alive (13.4%). Based on multivariate logistic regression, in our cohort of elderly patients undergoing non-cardiac surgery, moderate/severe preoperative anaemia (aOR 1.61, p = 0.04) and high preoperative RDW levels in the 3rd quartile (>13.4% and ≤14.3%) and 4th quartile (>14.3%) were significantly associated with increased odds of 30-day mortality - (aOR 2.12, p = 0.02) and (aOR 2.85, p = 0.001) respectively, after adjusting for the effects of transfusion, surgical severity, priority of surgery, and comorbidities. Patients with high RDW, defined as >15.7% (90th centile), and preoperative anaemia have higher odds of 30-day mortality compared to patients with anaemia and normal RDW. Thus, preoperative RDW independently increases risk of 30-day postoperative mortality, and future risk stratification strategies should include RDW as a factor

Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury

Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans

Cellular therapies for treating pain associated with spinal cord injury

Spinal cord injury leads to immense disability and loss of quality of life in human with no satisfactory clinical cure. Cell-based or cell-related therapies have emerged as promising therapeutic potentials both in regeneration of spinal cord and mitigation of neuropathic pain due to spinal cord injury. This article reviews the various options and their latest developments with an update on their therapeutic potentials and clinical trialing

A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words

Persistent left superior vena cava: Review of the literature, clinical implications, and relevance of alterations in thoracic central venous anatomy as pertaining to the general principles of central venous access device placement and venography in cancer patients

Persistent left superior vena cava (PLSVC) represents the most common congenital venous anomaly of the thoracic systemic venous return, occurring in 0.3% to 0.5% of individuals in the general population, and in up to 12% of individuals with other documented congential heart abnormalities. In this regard, there is very little in the literature that specifically addresses the potential importance of the incidental finding of PLSVC to surgeons, interventional radiologists, and other physicians actively involved in central venous access device placement in cancer patients. In the current review, we have attempted to comprehensively evaluate the available literature regarding PLSVC. Additionally, we have discussed the clinical implications and relevance of such congenital aberrancies, as well as of treatment-induced or disease-induced alterations in the anatomy of the thoracic central venous system, as they pertain to the general principles of successful placement of central venous access devices in cancer patients. Specifically regarding PLSVC, it is critical to recognize its presence during attempted central venous access device placement and to fully characterize the pattern of cardiac venous return (i.e., to the right atrium or to the left atrium) in any patient suspected of PLSVC prior to initiation of use of their central venous access device