Composite Resins Impregnated by Phosphorus Organic Extractants for Separation of Rare Earth Elements from Nitrate-Based Leachate of Permanent Magnets.

Composite resins impregnated by different organophosphorus extractants were developed and used for the extraction chromatography recovery of rare earth elements from nitrate-based leachate of NdFeB permanent magnets. The influence of different factors on recovery of Nd(III) and Fe(III), as the most difficult to separate elements, by developed resins was studied. The influence of extractant structure, the composition of feed solutions, and concentrations of HNO3 and NH4NO3 on the recovery of Fe(III) and Nd(III) by prepared resins were considered. The best recovery of Nd(III) was shown by resin impregnated with N,N-dioctyl (diphenylphosphoryl) acetamide. For this material, sorption characteristics (values of the distribution coefficient, capacity, and the Nd(III)/Fe(III) separation factor) were obtained, and the reproducibility of the loading-stripping process was evaluated. This resin and its precursors were characterized by IR spectroscopy. It was found that the developed resin is more efficient for Nd(III) recovery than resin impregnated with TODGA. An effective approach to the Nd(III)/Fe(III) separation with developed resin in nitrate solution was proposed. This approach was used for recovery of Pr(III), Nd(III), and Dy(III) from the nitrate-based leachate of NdFeB magnets by the developed resin. The final product contained 99.6% of rare earths.

Interleukin-4 Reduces Lesion Volume and Improves Neurological Function in the Acute Phase after Experimental Traumatic Brain Injury in Mice.

Little is known about the impact of interleukin-4 (IL-4) on secondary brain damage in the acute phase after experimental traumatic brain injury (TBI). Therefore, we evaluated the effect of IL-4-Knockout (IL-4-KO) on structural damage, as well as functional impairment, in the acute phase after experimental TBI in mice. A total of 28 C57Bl/6 wildtype and 20 C57BL/6-Il4tm1Nnt/J IL-4-KO mice were subjected to controlled cortical impact (CCI). Contusion volumes, body weight and functional outcome (Video Open Field Test [VOF], Hole Board Test [HB], CatWalkXT®) were determined on post-operative Days 1 (D1), 3 (D3), and 7 (D7). Contusion volume (13.45 ± 0.88 mm3 vs. 9.50 ± 0.97 mm3, p = 0.015) and weight loss (-2.92 ± 0.52% vs. -0.85 ± 0.67%, p = 0.027) were significantly higher and exploration behavior significantly more impaired (e.g., 150.44 ± 18.71 fields explored vs. 211.56 ± 18.90 fields explored, p = 0.028 in the VOF; 23.31 ± 2.03 holes explored vs. 35.65 ± 1.93 holes explored, p < 0.001 in the HB) in IL-4-KO mice on D1. Gait impairment was significantly more pronounced in IL-4-KO mice throughout the first week after CCI (e.g., 0.07 ± 0.01 sec vs. 0.00 ± 0.01 sec, p = 0.047 for right hindpaw Swing on D1; -1.76 ± 1.34 U vs. 2.53 ± 0.90 U, p = 0.01 for right forepaw mean intensity on D3; -0.01 ± 0.01 cm2 vs. 0.05 ± 0.01 cm2, p = 0.015 for left forepaw mean area on D7). In conclusion, IL-4 reduces structural damage and improves functional outcome in the acute phase after CCI. Neurobehavioral outcome assessment in IL-4-related studies should focus on motor function on the first 3 days after trauma induction.

Solvent-Impregnated Resins Based on the Mixture of (2-Diphenylphosphoryl)-4-ethylphenoxy)methyl) diphenylphosphine Oxide and Ionic Liquid for Nd(III) Recovery from Nitric Acid Media.

Novel solvent-impregnated resins (SIRs) were prepared by treatment of styrene-divinylbenzene copolymer (LPS-500) with mixtures of the promising polydentante extractant (2-diphenylphosphoryl)-4-ethylphenoxy)methyl)diphenylphosphine oxide (L) and an ionic liquid [C4mim]+[Tf2N]-for the extraction chromatography recovery of Nd(III) from nitric acid solutions. It was shown that introduction of the ionic liquid into the SIR composition results in considerable enhancement of the Nd(III) recovery efficiency compared with resin impregnated only by L in slightly acidic media. The influence of the L: ionic liquid molar ratio in the SIRs composition, their percentages, concentration of metal and HNO3 in the eluent, and acid type on the value of synergistic effect and adsorption efficiency of Nd(III) recovery was studied. The SIR containing 40% of mixture of L and [C4mim]+[Tf2N]- with molar ratio 2:1 turned out to be the most efficient. The selectivity of Nd(III) separation from light and heavy rare-earth elements was studied and the optimal conditions of Nd(III) adsorption recovery and stripping by this SIR were chosen. It was found that in recovery efficiency of Nd(III) developed SIR exceeded the SIR containing Cyanex 923 (a mixture of monodentate trialkylphosphine oxides) and [C4mim]+[Tf2N]-.

The miR-181a-SFRP4 Axis Regulates Wnt Activation to Drive Stemness and Platinum Resistance in Ovarian Cancer.

Wnt signaling is a major driver of stemness and chemoresistance in ovarian cancer, yet the genetic drivers that stimulate its expression remain largely unknown. Unlike other cancers, mutations in the Wnt pathway are not reported in high-grade serous ovarian cancer (HGSOC). Hence, a key challenge that must be addressed to develop effective targeted therapies is to identify nonmutational drivers of Wnt activation. Using an miRNA sensor-based approach, we have identified miR-181a as a novel driver of Wnt/ß-catenin signaling. miR-181ahigh primary HGSOC cells exhibited increased Wnt/ß-catenin signaling, which was associated with increased stem-cell frequency and platinum resistance. Consistent with these findings, inhibition of ß-catenin decreased stem-like properties in miR-181ahigh cell populations and downregulated miR-181a. The Wnt inhibitor SFRP4 was identified as a novel target of miR-181a. Overall, our results demonstrate that miR-181a is a nonmutational activator of Wnt signaling that drives stemness and chemoresistance in HGSOC, suggesting that the miR-181a-SFRP4 axis can be evaluated as a novel biomarker for ß-catenin-targeted therapy in this disease. SIGNIFICANCE: These results demonstrate that miR-181a is an activator of Wnt signaling that drives stemness and chemoresistance in HGSOC and may be targeted therapeutically in recurrent disease.

Compassionate Use of Remdesivir in Pregnant Women With Severe Coronavirus Disease 2019.

BACKGROUND: Remdesivir is efficacious for severe coronavirus disease 2019 (COVID-19) in adults, but data in pregnant women are limited. We describe outcomes in the first 86 pregnant women with severe COVID-19 who were treated with remdesivir. METHODS: The reported data span 21 March to 16 June 2020 for hospitalized pregnant women with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection and room air oxygen saturation ≤94% whose clinicians requested remdesivir through the compassionate use program. The intended remdesivir treatment course was 10 days (200 mg on day 1, followed by 100 mg for days 2-10, given intravenously). RESULTS: Nineteen of 86 women delivered before their first dose and were reclassified as immediate "postpartum" (median postpartum day 1 [range, 0-3]). At baseline, 40% of pregnant women (median gestational age, 28 weeks) required invasive ventilation, in contrast to 95% of postpartum women (median gestational age at delivery 30 weeks). By day 28 of follow-up, the level of oxygen requirement decreased in 96% and 89% of pregnant and postpartum women, respectively. Among pregnant women, 93% of those on mechanical ventilation were extubated, 93% recovered, and 90% were discharged. Among postpartum women, 89% were extubated, 89% recovered, and 84% were discharged. Remdesivir was well tolerated, with a low incidence of serious adverse events (AEs) (16%). Most AEs were related to pregnancy and underlying disease; most laboratory abnormalities were grade 1 or 2. There was 1 maternal death attributed to underlying disease and no neonatal deaths. CONCLUSIONS: Among 86 pregnant and postpartum women with severe COVID-19 who received compassionate-use remdesivir, recovery rates were high, with a low rate of serious AEs.

The CatWalk XT® is a valid tool for objective assessment of motor function in the acute phase after controlled cortical impact in mice.

BACKGROUND: Despite multiple experimental models of traumatic brain injury (TBI) being available, objective assessment of gait and motor function in rodents remains difficult; therefore, we studied the value of the CatWalk XT® gait analysis as an observer independent, automated method for outcome assessment in one of the most frequently used preclinical TBI model, the controlled cortical impact (CCI), in mice. METHODS: C57Bl/6 mice were subjected to either right parietal CCI or a sham procedure. Functional outcome was assessed using the CatWalk XT® (Version 10.6) as well as the hole board test at days one, three and seven after trauma induction. RESULTS: CCI led to diffuse, asymmetric and bilateral disturbances of both static and dynamic parameters in the CatWalk XT® gait analysis. The CatWalk XT® detected even minimal but statistically significant impairments that could not have been detected by clinical assessments. Impairments of static parameters were most pronounced within the first three days and diminished thereafter, while dynamic parameters were impaired until seven days after CCI. Fittingly, mice explored the hole board significantly less on day three trauma induction. CONCLUSIONS: The CatWalk XT® is a valid tool for objective assessment of motor function in the acute phase after controlled cortical impact TBI in mice. Similar to observations made in humans, CCI leads to impairments of both static and dynamic parameters of gait and motor function which persist throughout the first week after the injury.

Hyperhomocysteinemia and Pulmonary Embolism in a Young Male.

The association of hyperhomocysteinemia with thrombosis has provoked debate in the medical literature. Although studies have found associations between moderate homocysteine elevations and thrombotic events, others dispute this relationship. We present herein the case of a 24-year-old male who presented with unprovoked bilateral submassive pulmonary emboli. Extensive hypercoagulability workup was notable for an elevated homocysteine level, in addition to low vitamin B12 and folate levels. Of note, the patient had a history of small bowel resection after trauma, which may have contributed to the aforementioned metabolic derangements, potentially increasing his risk for thrombosis and interfering with the efficacy of his anticoagulation.

Cognitive disorders in persons of working age with dyscirculatory encephalopathy.

OBJECTIVE: The aim of the study was to study the neuropsychological features of cerebrovascular disorders in persons of working age at the outpatient clinic stage. PATIENTS AND METHODS: Materials and methods: 90 persons of working age were surveyed. Clinical neurological and clinical instrumental examination. RESULTS: Results: The first group consisted of patients with dyscirculatory encephalopathy on the background of arterial hypertension - 60 people, the second with dyscirculatory encephalopathy on the background of cerebral atherosclerosis - 30. Assessment of cognitive function was examined using the MMSE Mental Status Scale, Anxiety, and Depression Scale using the DASS-21 scale. CONCLUSION: Conclusions: According to the results of the study, significant memory impairment on the MMSE scale (p <0.003) was found in patients with dyscirculatory encephalopathy without arterial hypertension. In patients with dyscirculatory encephalopathy on the background of hypertension, anxiety (p <0.001) and depressive disorders (p = 0.033) were significantly more prevalent compared with patients with dyscirculatory encephalopathy without arterial hypertension. The increase in depressive disorders and anxiety in patients with dyscirculatory encephalopathy on the background of arterial hypertension decreases orientation and memory.

The Effect of Forced Cough to Minimize Pain and Discomfort at the Time of Colposcopy-Guided Cervical Biopsy.

OBJECTIVE: The aim of the study was to determine whether forced cough during colposcopy-guided cervical biopsy affected pain and anxiety levels. MATERIALS AND METHODS: The study was conducted at the University Hospital (Newark, NJ) Ambulatory Care Center from December 2016 to June 2018 and evaluated 110 patients at the time of a colposcopy-guided cervical biopsy. Study patients were randomized to either cough or no cough group during the biopsy procedure. Pain level was assessed using a visual analog pain scale before, during, and immediately after a colposcopy-guided cervical biopsy. Study patients also completed a standardized anxiety survey before and after the procedure. T tests, Pearson χ, or Cochran-Mantel-Haenszel were used to compare baseline characteristics between the cough and no cough groups. Multivariate linear regression analysis was used to identify potential confounders and then compare pain levels across both groups. RESULTS: There was no statistically significant difference in pain scores between the cough and no cough group when analyzed for each demographic variable even when confounders were accounted for. The anxiety scores for both study groups before and after the procedure were similar and not significantly reduced. CONCLUSIONS: We observed a trend that cough reduced pain associated with the colposcopy-guided cervical biopsy but did not reach statistical significance. A similar outcome was observed in anxiety level, where anxiety was reduced in the cough group but was not statistically significant as compared with the no cough group. Further studies are necessary to assess various modalities in reducing pain and anxiety associated with colposcopy-guided cervical biopsy.

Evaluating class III antiarrhythmic agents as novel MYC targeting drugs in ovarian cancer.

OBJECTIVE: To evaluate the utility of amiodarone and its derivative dronedarone as novel drug repositioning candidates in EOC and to determine the potential pathways targeted by these drugs. METHODS: Drug-predict bioinformatics platform was used to assess the utility of amiodarone as a novel drug-repurposing candidate in EOC. EOC cells were treated with amiodarone and dronedarone. Cell death was assessed by Annexin V staining. Cell viability and cell survival were assessed by MTT and clonogenics assays respectively. c-MYC and mTOR/Akt axis were evaluated as potential targets. Effect on autophagy was determined by autophagy flux flow cytometry. RESULTS: "DrugPredict" bioinformatics platform ranked Class III antiarrhythmic drug amiodarone within the top 3.9% of potential EOC drug repositioning candidates which was comparable to carboplatin ranking in the top 3.7%. Amiodarone and dronedarone were the only Class III antiarrhythmic drugs that decreased the cellular survival of both cisplatin-sensitive and cisplatin-resistant primary EOC cells. Interestingly, both drugs induced degradation of c-MYC protein and decreased the expression of known transcriptional targets of c-MYC. Furthermore, stable overexpression of non-degradable c-MYC partially rescued the effects of amiodarone and dronedarone induced cell death. Dronedarone induced higher autophagy flux in EOC cells as compared to amiodarone with decreased phospho-AKT and phospho-4EBP1 protein expression, suggesting autophagy induction due to inhibition of AKT/mTOR axis with these drugs. Lastly, both drugs also inhibited the survival of EOC tumor-initiating cells (TICs). CONCLUSIONS: We provide the first evidence of class III antiarrhythmic agents as novel c-MYC targeting drugs and autophagy inducers in EOC. Since c-MYC is amplified in >40% ovarian tumors, our results provide the basis for repositioning amiodarone and dronedarone as novel c-MYC targeting drugs in EOC with potential extension to other cancers.

Mitotic Exit Dysfunction through the Deregulation of APC/C Characterizes Cisplatin-Resistant State in Epithelial Ovarian Cancer.

Purpose: Acquired resistance to cisplatin is a major barrier to success in treatment of various cancers, and understanding mitotic mechanisms unique to cisplatin-resistant cancer cells can provide the basis for developing novel mitotic targeted therapies aimed at eradicating these cells.Experimental Design: Using cisplatin-resistant models derived from primary patient epithelial ovarian cancer (EOC) cells, we have explored the status of mitotic exit mechanisms in cisplatin-resistant cells.Results: We have uncovered an unexpected role of long-term cisplatin treatment in inducing mitotic exit vulnerability characterized by increased spindle checkpoint activity and functional dependency on Polo-like kinase 1 (PLK1) for mitotic exit in the presence of anaphase promoting complex/cyclosome (APC/C) dysfunction in a cisplatin-resistant state. Accordingly, PLK1 inhibition decreased the survival of cisplatin-resistant cells in vitro and in vivo and exacerbated spindle checkpoint response in these cells. APC/CCDC20 inhibition increased sensitivity to pharmacologic PLK1 inhibition, further confirming the existence of APC/C dysfunction in cisplatin-resistant cells. In addition, we uncovered that resistance to volasertib, PLK1 inhibitor, is due to maintenance of cells with low PLK1 expression. Accordingly, stable PLK1 downregulation in cisplatin-resistant cells induced tolerance to volasertib.Conclusions: We provide the first evidence of APC/C dysfunction in cisplatin-resistant state, suggesting that understanding APC/C functions in cisplatin-resistant state could provide a basis for developing novel mitotic exit-based therapies to eradicate cisplatin-resistant cancer cells. Our results also show that PLK1 downregulation could underlie emergence of resistance to PLK1-targeted therapies in cancers. Clin Cancer Res; 24(18); 4588-601. ©2018 AACR.

Critical role of Wnt/ß-catenin signaling in driving epithelial ovarian cancer platinum resistance.

Resistance to platinum-based chemotherapy is the major barrier to treating epithelial ovarian cancer. To improve patient outcomes, it is critical to identify the underlying mechanisms that promote platinum resistance. Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells, but are unable to eliminate cancer initiating cells (CIC). To date, the relevant pathways that regulate ovarian CICs remain elusive. Several correlative studies have shown that Wnt/ß-catenin pathway activation is associated with poor outcomes in patients with high-grade serous ovarian cancer (HGSOC). However, the functional relevance of these findings remain to be delineated. We have uncovered that Wnt/ß-catenin pathway activation is a critical driver of HGSOC chemotherapy resistance, and targeted inhibition of this pathway, which eliminates CICs, represents a novel and effective treatment for chemoresistant HGSOC. Here we show that Wnt/ß-catenin signaling is activated in ovarian CICs, and targeted inhibition of ß-catenin potently sensitized cells to cisplatin and decreased CIC tumor sphere formation. Furthermore, the Wnt/ß-catenin specific inhibitor iCG-001 potently sensitized cells to cisplatin and decreased stem-cell frequency in platinum resistant cells. Taken together, our data is the first report providing evidence that the Wnt/ß-catenin signaling pathway maintains stem-like properties and drug resistance of primary HGSOC PDX derived platinum resistant models, and therapeutic targeting of this pathway with iCG-001/PRI-724, which has been shown to be well tolerated in Phase I trials, may be an effective treatment option.

Maternal and fetoplacental hypoxia do not alter circulating angiogenic growth effectors during human pregnancy.

One causal model of preeclampsia (PE) postulates that placental hypoxia alters the production of angiogenic growth effectors (AGEs), causing an imbalance leading to maternal endothelial cell dysfunction. We tested this model using the natural experiment of high-altitude (HA) residence. We hypothesized that in HA pregnancies 1) circulating soluble fms-like tyrosine kinase 1 (sFlt-1) is increased and placental growth factor (PlGF) decreased, and 2) AGE concentrations correlate with measures of hypoxia. A cross-sectional study of healthy pregnancies at low altitude (LA) (400 m) versus HA (3600 m) compared normal (n = 80 at HA, n = 90 at LA) and PE pregnancies (n = 20 PE at HA, n = 19 PE at LA). Blood was collected using standard serum separation and, in parallel, by a method designed to inhibit platelet activation. AGEs were measured by enzyme-linked immunosorbent assays. AGEs did not differ between altitudes in normal or PE pregnancies. AGE concentrations were unrelated to measures of maternal or fetal hypoxia. PlGF was lower and sFlt-1 higher in PE, but overlapped considerably with the range observed in normal samples. PlGF correlated with placental mass in both normal and PE pregnancies. The contribution of peripheral cells to the values measured for AGEs was similar at LA and HA, but was greater in PE than in normotensive women. Hypoxia, across a wide physiological range in pregnancy, does not alter levels of circulating AGEs in otherwise normal pregnancies. Peripheral cell release of AGEs with the hemostasis characteristic of standard blood collection is highly variable and contributes to a doubling of the amount of sFlt-1 measured in PE as compared to normal pregnancies.

Intrinsic mitochondrial DNA repair defects in Ataxia Telangiectasia.

Ataxia Telangiectasia (A-T) is a progressive childhood disorder characterized most notably by cerebellar degeneration and predisposition to cancer. A-T is caused by mutations in the kinase ATM, a master regulator of the DNA double-strand break response. In addition to DNA-damage signaling defects, A-T cells display mitochondrial dysfunction that is thought to contribute to A-T pathogenesis. However, the molecular mechanism leading to mitochondrial dysfunction in A-T remains unclear. Here, we show that lack of ATM leads to reduced mitochondrial DNA (mtDNA) integrity and mitochondrial dysfunction, which are associated to defective mtDNA repair. While protein levels of mtDNA repair proteins are essentially normal, in the absence of ATM levels specifically of DNA ligase III (Lig3), the only DNA ligase working in mitochondria is reduced. The reduction of Lig3 is observed in different A-T patient cells, in brain and pre-B cells derived from ATM knockout mice as well as upon transient or stable knockdown of ATM. Furthermore, pharmacological inhibition of Lig3 in wild type cells phenocopies the mtDNA repair defects observed in A-T patient cells. As targeted deletion of LIG3 in the central nervous system causes debilitating ataxia in mice, reduced Lig3 protein levels and the consequent mtDNA repair defect may contribute to A-T neurodegeneration. A-T is thus the first disease characterized by diminished Lig3.

Human umbilical-cord-blood mononucleated cells enhance the survival of lethally irradiated mice: dosage and the window of time.

The purpose of this study was to evaluate the window of time and dose of human umbilical-cord-blood (HUCB) mononucleated cells necessary for successful treatment of radiation injury in mice. Female A/J mice (27-30 weeks old) were exposed to an absorbed dose of 9-10 Gy of (137)Cs γ-rays delivered acutely to the whole body. They were treated either with 1 × 10(8) or 2 × 10(8) HUCB mononucleated cells at 24-52 h after the irradiation. The antibiotic Levaquin was applied 4 h postirradiation. The increased dose of cord-blood cells resulted in enhanced survival. The enhancement of survival in animals that received 2 × 10(8) HUCB mononucleated cells relative to irradiated but untreated animals was highly significant (P < 0.01). Compared with earlier studies, the increased dose of HUCB mononucleated cells, coupled with early use of an antibiotic, extended the window of time for effective treatment of severe radiation injury from 4 to 24-52 h after exposure.

Expression of (NES-)hTERT in cancer cells delays cell cycle progression and increases sensitivity to genotoxic stress.

Telomerase is a reverse transcriptase associated with cellular immortality through telomere maintenance. This enzyme is activated in 90% of human cancers, and inhibitors of telomerase are currently in clinical trials to counteract tumor growth. Many aspects of telomerase biology have been investigated for therapy, particularly inhibition of the enzyme, but little was done regarding its subcellular shuttling. We have recently shown that mutations in the nuclear export signal of hTERT, the catalytic component of telomerase, led to a mutant ((NES-)hTERT) that failed to immortalize cells despite nuclear localization and catalytic activity. Expression of (NES-)hTERT in primary fibroblast resulted in telomere-based premature senescence and mitochondrial dysfunction. Here we show that expression of (NES-)hTERT in LNCaP, SQ20B and HeLa cells rapidly and significantly decreases their proliferation rate and ability to form colonies in soft agar while not interfering with endogenous telomerase activity. The cancer cells showed increased DNA damage at telomeric and extra-telomeric sites, and became sensitive to ionizing radiation and hydrogen peroxide exposures. Our data show that expression of (NES-)hTERT efficiently counteracts cancer cell growth in vitro in at least two different ways, and suggest manipulation with the NES of hTERT or its subcellular shuttling as a new strategy for cancer treatment.

The effect of human cord blood therapy on the intestinal tract of lethally irradiated mice: possible use for mass casualties.

PURPOSE: To evaluate the recovery of the gastrointestinal tract in lethally irradiated mice treated with human cord blood and antibiotics. MATERIALS AND METHODS: A/J mice were randomly assigned to seven study groups, including groups exposed to acute 9 Gy from (137)Cs gamma-rays to the whole body. Four hours after irradiation, exposed mice were treated with either cord blood nucleated cells, Levaquin, or a combination of both. Weight gain/loss and survival were monitored for 2 months. Upon death or euthanasia, the organs were prepared for molecular and histological analyses. RESULTS: Whereas irradiated mice (n = 9) lived 6-15 days, approximately 60% of irradiated mice that received the combined treatment (n = 7) survived more than 50 days. None of the treated animals developed Graft versus Host disease. All animals lost weight after irradiation; however, the 50(+) days-survivors (n = 4) gained on average approximately 1.8 g over their initial weight. Whereas hemorrhagic bone marrow and large areas of transmural necrosis were observed in the bowel of the irradiated mice, the 50(+) days-survivors showed recovery of the bone marrow. They behaved normally and had significant but incomplete recovery of the intestinal and colonic mucosa. Human DNA was detected in their organs, particularly in the large intestine. CONCLUSION: Red cell-depleted cord blood transfusions combined with antibiotic treatment contribute to bone marrow and gastrointestinal recovery in high dose-irradiated mice, and may be an available therapy for mass casualties during radiological emergencies.

A mutant telomerase defective in nuclear-cytoplasmic shuttling fails to immortalize cells and is associated with mitochondrial dysfunction.

Telomerase is a reverse transcriptase specialized in telomere synthesis. The enzyme is primarily nuclear where it elongates telomeres, but many reports show that the catalytic component of telomerase (in humans called hTERT) also localizes outside of the nucleus, including in mitochondria. Shuttling of hTERT between nucleus and cytoplasm and vice versa has been reported, and different proteins shown to regulate such translocation. Exactly why telomerase moves between subcellular compartments is still unclear. In this study we report that mutations that disrupt the nuclear export signal (NES) of hTERT render it nuclear but unable to immortalize cells despite retention of catalytic activity in vitro. Overexpression of the mutant protein in primary fibroblasts is associated with telomere-based cellular senescence, multinucleated cells and the activation of the DNA damage response genes ATM, Chk2 and p53. Mitochondria function is also impaired in the cells. We find that cells expressing the mutant hTERT produce high levels of mitochondrial reactive oxygen species and have damage in telomeric and extratelomeric DNA. Dysfunctional mitochondria are also observed in an ALT (alternative lengthening of telomeres) cell line that is insensitive to growth arrest induced by the mutant hTERT showing that mitochondrial impairment is not a consequence of the growth arrest. Our data indicate that mutations involving the NES of hTERT are associated with defects in telomere maintenance, mitochondrial function and cellular growth, and suggest targeting this region of hTERT as a potential new strategy for cancer treatment.

Analysis of oxidative damage by gene-specific quantitative PCR.

This unit describes the gene-specific quantitative PCR-based (QPCR) assay, which is used to measure DNA integrity of both nuclear and mitochondrial genomes based on amplification of long DNA targets. QPCR can be used to quantify the formation of DNA damage and the kinetics of DNA repair by following restoration of amplification of the target DNA over time after removal of the damaging agent. A detailed protocol to set up QPCR in any laboratory, highlighting critical parameters for successful establishment of the assay and interpretation of the results, is provided here. Advantages (e.g., the use of nanogram amounts of DNA) and limitations (e.g., the inability to define the specific type of lesion present on the DNA) of using QPCR to assay DNA damage in human cells are also described.