Analysis Of Telomerase Activity Research Articles

Single-molecule detection assisted by the target-triggered signal amplification strategy for ultrasensitive quantitative analysis of intracellular telomerase activity.

We developed a multiplex single-molecule quantitative assay of intracellular telomerase that used target-triggered signal amplification to enhance sensitivity, substrate reaction to increase signal stability, and quantum dots to enhance signal-to-noise ratio, obtaining an LOD of 5 × 10-14 IU for intracellular telomerase and LOD of 3 cells for multiple cancer cells.

An Activatable Nanoscintillator Probe for Detecting Telomerase Activity and Screening Inhibitors In Vivo

Telomerase represents an essential molecular machinery for tumor occurrence and progression and a potential therapeutic target for cancer treatment. Sensitive and reliable analysis of telomerase activity is of significant importance for the diagnosis and treatment of cancer. In this study, we developed a telomerase-activated nanoscintillator probe for deep-tissue and background-free imaging of telomerase activity and screening telomerase inhibitors in tumor-bearing living mice models. The probe was constructed by modifying lanthanide-doped nanoscintillators with aptamer-containing DNA anchor strands which hybridized with quencher labelled–oligonucleotide strands and telomerase primers. The X-ray-induced fluorescence of the probe was quenched originally but turned on upon telomerase-catalyzed extension of the primer. Benefiting from exceptional tissue penetrating properties and negligible autofluorescence of X-ray excitation, this probe enabled direct detection of telomerase activity in vivo via fluorescence imaging. Furthermore, with the direct, readable fluorescent signals, the probe enabled the screening of telomerase inhibitors in living cells and whole-animal models in the native states of telomerase. This strategy would inspire the development of low autofluorescence and deep tissue bioimaging probes for disease diagnosis and drug development in high-level living settings.

Biosensing of telomerase antigen using sandwich type immunosensor based on poly(β-Cyclodextrin) decorated by Au@Pt nanoparticles: An innovative immune-platform toward early-stage identification of cancer

Telomeres maintain genomic integrity in normal cells and their gradual shortening during successive cell divisions causes chromosomal instability. Sensitive and reliable analysis of telomerase activity is essential for clinical diagnosis of cancer. In this study, a novel sandwich type and label-free electrochemical immunosensor was developed for the efficient monitoring of telomerase antigen based on Au@Pt nano-alloy and Poly (beta-Cyclodextrin (β-CD)) functionalized with AuNPs-DDT and toluidine blue. For the first time, KCC-1-NH-CS2 was used as an advanced substrate to encapsulation of biotinylated antibody (Ab1) of telomerase towards maintain of antibody in the stress condition of solution. By providing a wide surface to volume using dendritic fibrous Nanosilica for the effective immobilization of the biotinylated antibody, a sensitive immunosensor was designed for the probing of antigen–antibody immunocomplex. Interestingly, the P(β-CD) assured the bioactivity and stability of the immobilized 2-AG antibody. To complete the capping part of the immunosensor (reporter), an HRP-conjugated antibody of telomerase (secondary antibody (Ab2)) was coupled to the surface of a gold electrode (AuE) modified by P(β-CD), as well as a predominantly biotinylated antibody (Ab1, encapsulated into the KCC-1-NH-CS2). Engineered immune-platform demonstrated acceptable analytical properties (LLOQ = 0.078 IU.ml). In addition, the created biosensor shows excellent selectivity and acceptable ability for the detection of telomerase in human plasma samples with satisfactory stability which might hold great application potential in cancer analysis and clinical diagnosis. This research inspires scientists and researchers to generate and develop novel biosensor platforms for point-of-care (POC) diagnostics of telomerase. Based on accurate, sensitive, inexpensive, and time-saving futures of developed biosensor compared to other methods makes it a useful platform for future analytical works.

Telomerase activity, relative telomere length, and longevity in alfalfa (Medicago sativa L.)

Background Medicago sativa L. ‘Qingshui’ is a valuable rhizomatous forage germplasm resource. We previously crossed Qingshui with the high-yielding Medicago sativa L. ‘WL168’ and obtained novel rhizomatous hybrid strains (RSA-01, RSA-02, and RSA-03). Telomere dynamics are more accurate predictors of survival and mortality than chronological age. Based on telomere analyses, we aimed to identify alfalfa varieties with increased stamina and longevity for the establishment of artificial grazing grasslands. Methods In this study, we performed longitudinal analysis of telomerase activity and relative telomere length in five alfalfa varieties (Qingshui, WL168, RSA-01, RSA-02, and RSA-03) at the age of 1 year and 5 years to examine the relationship among telomerase activity, rate of change in relative telomere length, and longevity. We further aimed to evaluate the longevity of the examined varieties. Telomerase activity and relative telomere length were measured using enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. Results We observed significant differences in telomerase activity between plants aged 1 year and those aged 5 years in all varieties except WL168, and the rate of change in telomerase activity does not differ reliably with age. As telomerase activity and relative telomere length are complex phenomena, further studies examining the molecular mechanisms of telomere-related proteins are needed. Relative telomere lengths of Qingshui, WL168, RSA-01, RSA-02, and RSA-03 in plants aged 5 years were higher than those aged 1 year by 11.41, 11.24, 9.21, 10.23, and 11.41, respectively. Relative telomere length of alfalfa tended to increase with age. Accordingly, alfalfa varieties can be classified according to rate of change in relative telomere length as long-lived (Qingshui, WL168, and RSA-03), medium-lived (RSA-02) and short-lived (RSA-01). The differences in relative telomere length distances of Qingshui, WL168, RSA-01, RSA-02, and RSA-03 between plants aged 1 and 5 years were 10.40, 13.02, 12.22, 11.22, and 13.25, respectively. The largest difference in relative telomere length was found between Qingshui and RSA-02 at 2.20. Our findings demonstrated that relative telomere length in alfalfa is influenced by genetic variation and age, with age exerting a greater effect.

A dual-amplification label-free ratiometric fluorescent sensor for accurate monitoring of telomerase activity based on unique intercalation characteristics of dyes toward different DNA structures

Accurate evaluation and reliable quantitation of telomerase activity in various cancer cells is of significant importance to early cancer diagnosis and therapy. All previously reported ratiometric fluorescence telomerase activity assays inevitably required the expensive labeling or sophisticated probe synthesis, which were laborious and time-consuming. To overcome such restrictions, herein we developed, for the first time, a facile and label-free ratiometric fluorescence sensing strategy for accurately and sensitively detecting telomerase activity based on unique intercalation characteristics of dyes toward double-stranded DNA and G-quadruplex, respectively. By taking advantage of the dual amplification method consisting of catalytic hairpin assembly and exonuclease III assisted cleavage reaction, telomerase activity can be quantitatively detected in MCF-7 cells ranging from 50 to 2000 with a detection limit of 16 MCF-7 cells. Furthermore, our approach could distinguish the relative telomerase activity in cancer cells from that in normal cells, as well as in different cancer cells. It was also confirmed that our strategy displayed good analytical performance for screening the inhibitors for telomerase. More importantly, compared to those labeling and/or single signal-based methods for telomerase activity detection, this assay not only effectively circumvented the expensive labeling and complex probe preparation but also avoided false-positive results, thus exhibiting the appealing features of simplicity, low cost, and high accuracy. Overall, our method might be a promising tool in telomerase activity analysis and telomerase-targeted antitumor drugs screening.

Aptamer-engineered gold nanorod driven an absorbance enhanced strategy for sensitive biomacromolecule profiling

Gold nanorods (AuNRs)-based plasmonic biosensor offers new opportunity for quantification of biomacromolecules due to their high designability and low technical demands. However, existing methods for the optical detection of biomacromolecule require the targets to induce the aggregation or etching of AuNRs. This limits the range of targets that can be detected, because molecules at extremely low concentration are difficult to arouse aggregation or etching of AuNRs. Thus, it is still challenge to design a scheme for the biomacromolecules at extremely low concentration which can't arouse aggregation or etching of AuNRs based on their plasmonic property. This study proposes a universal absorbance enhanced strategy for biomacromolecule detection with aptamers engineered AuNRs. The biosensor assay (Apts/AuNRs) is designed through assembly of two aptamers on AuNRs to specified recognize the target biomacromolecules, forming closed-loop conformation based on the proximity-dependent ligation, producing absorbance enhancement in the plasmonic peak of AuNRs. It is interesting that the absorbance enhancement increases gradually with increasing protein concentration within a certain range, whereas no aggregation or etching of AuNRs was observed compared with the typical AuNRs based LSPR sensor. Taking advantage of the excellent near infrared light absorption of AuNRs, Apts/AuNRs could be utilized to detect red protein such as cytochrome C, which exhibited better performance than AuNPs based plasmonic sensor. On this basis, the selectivity detection of cytochrome C with the detection of limit down to picomole level was demonstrated. By changing the type of aptamers on AuNRs, the sensitive and credible method was also utilized for the analysis of telomerase activity in nerve cell lysate. Telomerase activity in 4 × 104 neuroblastoma cell was determined to be about 3.575 U/L, which was close to the result of ELISA kit. Good recovery was achieved using standard samples recovery. This study broadens the scope of AuNRs based plasmonic ‬‬‬‬‬‬property ‬‬‬‬and offer a simple, sensitive and selective strategy for biomacromolecules detection in complexed biofluid.

Accurate Identification and Early Diagnosis of Osteosarcoma through CRISPR-Cas12a-Based Average Telomerase Activity Detection.

Sensitive and reliable analysis of telomerase activity is important for clinical diagnosis, therapy, and prognosis of osteosarcoma. Telomerase activity is a complicated concept including both the amount of active telomerases and the length of the telomerases extension product. Still, few of the strategies formerly proposed distinguish the two aspects of telomerase activity. Herein, we propose a novel CRISPR-Cas12a-based fluorescent sensing platform that can output signals of both the amounts of telomerase and length of telomerase extension products with the assistance of an elegantly designed stem-loop probe and CRISPR-Cas12a system. On this basis, we induced a novel index, average telomerase activity, for accurate cancer reporting. Through systematic laboratory and clinical experiments, we have demonstrated that average telomerase activity can accurately distinguish cancer cells and has the potential for osteosarcoma staging.

Association of cardiovascular risk factors with biological age

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Academy of Medical Sciences of Ukraine Background Identifying reliable biomarkers of early ageing is an important goal for the prevention of cardiovascular disease (CVD). Chronological age (CA) is an essential risk factor for age-related disease including CVD. Biological age (BA) may be a more accurate metric for the risk of CVD and prediction of early vascular ageing, atherosclerosis. Available data allow us to judge the relationship of early ageing with all-cause mortality, healthspan and CVD. There is a hypothesis that BA can be used into models of risk prediction and stratification of CVD for personalized treatment. Purpose to determine the relationship between BA and factors of risk CVD in patients of different ages without clinical manifestations CVD. Methods We selected 102 patients aged 31–60 years with moderate cardiovascular risk without clinical symptoms of CVD. The patients did not receive regular drug therapy. Consenting subjects had a physical assessment, anthropometric measurements, electrocardiogram recording, blood sampling for laboratory analyses, including analysis of telomerase activity, telomere length in leukocytes and buccal epithelium by a polymerase chain reaction in real-time. The SCORE scale was used for evaluation of the 10-year risk of fatal stroke and fatal myocardial infarction. In addition, 40 control individuals aged 31–60 years (20 men and 20 women), were also included in the study. BA was determined by 3 methods: PhenoAge, Voitenko’s method and Gorelkin-Pinhasov’s method. Multiple logistic regression analysis was used to develop a prediction of the early ageing model. Results The comparing patients of the control and main groups of the same CA shows, that patients with CVD risk have significantly higher BA, higher levels of proatherogenic lipids and shorter telomere length of the buccal epithelium. It was determined that body mass index, blood pressure, glucose levels are associated with an increase in BA in the main and the control groups. The increase of BA in the control group was associated with smoking, telomere length and telomerase activity and the level of antioxidant protection, in patients of the main group premature aging was associated with impaired lipid metabolism. Conclusion The biomarkers of biological aging can have benefits of the early identification of persons who age "faster" than others. The possibility of measuring biological aging can allow the assessment of health status at a time when there are still no symptoms, and interventions are more likely to be effective.

Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

BackgroundDerivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed.MethodsMonolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week.ResultsThe population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded.ConclusionsOur results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

Tetrahedral DNA Nanoconjugates for Simultaneous Measurement of Telomerase Activity and miRNA.

In this study, a tetrahedral DNA nanostructure was first self-assembled; this was then conjugated with gold nanoparticles (AuNPs) and carbon nanodots (CDs). The fabricated nanocomposites allow simultaneous analysis of telomerase activity and miRNA with dual fluorescence channels. By further introducing an iRGD peptide sequence, the nanoconjuates can be conveniently transferred inside living cells for in situ imaging. The analytical performances and anti-jamming capabilities are excellent. Meanwhile, the materials are highly biocompatible for intracellular applications. Therefore, the proposed biosystem shows great promise as a powerful tool for quantitative analysis of the dual biomarkers. The strategy can also be further exploited as a versatile platform for in situ detection of many other targets for early disease diagnosis.

Proximity binding-triggered multipedal DNA walker for the electrochemiluminescence detection of telomerase activity

The sensitive detection of telomerase activity is of great significance for the early diagnosis and treatment of cancer. Here, an innovative electrochemiluminescence resonance energy transfer (ECL-RET) sensor was explored to reliably detect telomerase activity based on proximity binding-triggered multipedal DNA walker. In this system, CdS quantum dots (CdS QDs) and silver nanoclusters (Ag NCs) were applied as ECL donor and acceptor, respectively. By ingeniously introducing a repetitive bases sequence (TTAGGG) along the telomerase primer, multiple same DNA “legs” were formed, leading to the activation of proximity binding-triggered multipedal DNA walker. Unlike the traditional unipedal DNA walker, one walking step of multipedal DNA walker concurrently initiated the responsivity of multiple signals, resulting in the shortening of the walking time and improvement of the signal amplification efficiency. Thus, under optimal conditions, the designed ECL-RET sensor exhibited a wide dynamic correlation of HeLa cells’ telomerase activity from 1 × 102 to 1 × 106 cells/mL and a low detection limit of 16 cells/mL. Moreover, this sensor realized the general and reliable analysis of telomerase activity in different cell lines. Due to the outstanding application potential in real samples, it is believed that the ECL-RET sensing system provides a new approach for the application of telomerase activity assays in cancer diagnostics.

Catalytic Hairpin Assembly-Assisted Rolling Circle Amplification for High-Sensitive Telomerase Activity Detection

Telomerase is a promising biomarker and a potential therapeutic target of malignant tumors. Reliable, facile, and sensitive telomerase activity analysis is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, we proposed a novel fluorescent assay termed catalytic hairpin assembly-assisted rolling circle amplification (CAR) for both in vitro and in situ high-sensitive telomerase activity detection. In the presence of active telomerase, the extension of a designed telomerase primer was limited to five bases (GGGTT), thus forming short telomerase products. Afterward, the obtained telomerase extension products cyclized Padlock and subsequently initiated the rolling circle amplification (RCA). In order to maintain a higher sensitivity, an ingeniously designed catalytic hairpin assembly (CHA) was attached for both signal amplification and result readout. The highlights of the CAR method were concluded as follows: (i) dual signal amplification from CHA and RCA ensures high sensitivity and (ii) the CAR method has the potential for both in vitro and intracellular imaging of telomerase activity. We believe that the CAR method would be of great potential for the diagnosis and therapy of various human diseases.

Highly sensitive electrochemical analysis of telomerase activity based on magnetic bead separation and exonuclease III-aided target recycling amplification

Telomerase is considered a pivotal biomarker for early cancer diagnosis and a valuable therapeutic target. However, the current methods to detect telomerase activity have some limitations. Herein, we propose a homogeneous electrochemical strategy to develop a simple, rapid, and highly sensitive assay to detect human telomerase activity from crude cancer cell extracts. Our strategy is based on magnetic bead separation and exonuclease III-aided target recycling amplification. The complementary probes can hybridize with the extended telomeric repeats, which allows exonuclease III to recognize and digest the latter once the hybrid product is separated with magnetic beads. The released complementary probes can hybridize with and open multiple methylene blue (MB)-labeled hairpin (HP) DNA probes, allowing exonuclease III to digest the duplex. Then, the opened hairpin couples with the captured mononucleotides on the surface of the gold electrode. By taking advantage of the exonuclease III-aided target recycling strategy, the present assay enables the detection of telomerase activity at a single-cell level. Furthermore, the assay is carried out in a homogeneous solution achieved by magnetic purification, which removes the interferents present in crude lysates and avoids false negatives, thus, providing a powerful platform to detect telomerase activity in samples of early-stage cancer.

Ratiometric Electrochemical Sensing Strategy for the Ultrasensitive Detection of Telomerase Activity

AbstractWe fabricated a ratiometric electrochemical strategy for the ultrasensitive analysis of telomerase activity. The hairpin DNA probe labeled with methylene blue (MB) tags at its 5’ termini is firstly immobilized on the gold electrode. It then hybridizes with the telomerase substrate primer labeled with ferrocene (Fc) tags. With the telomerase‐catalyzed reaction, a repeating sequence of (TTAGGG)n is generated, which is able to trigger the conformational transduction of the hairpin DNA probe. Consequently, MB tags are released from the electrode surface and the oxidation peak current (IMB) decreases. Meanwhile, the Fc signal remains stable and the peak current (IFc) can be used as the internal control, which improves the signal stability. This smart design allows reliable target recognition and the obtained results assure excellent analytical performances. IMB/IFc is found to be linearly related with logarithmic telomerase concentration with a quite low limit of detection (0.02 cells/μL). This proposed ratiometric method may emerge as a powerful practical tool for the analysis of telomerase activity, which may also be developed as a versatile tool for other nucleic acid assays.

FRET investigation toward DNA tetrahedron-based ratiometric analysis of intracellular telomerase activity.

Telomerase catalyzes the elongation of telomeres, which is closely associated with tumorigenesis. Therefore, development of reliable and convenient methods for telomerase activity analysis is critical for clinical diagnosis of cancers. However, most of the current probes for intracellular telomerase activity assay suffer from inevitable false-positive interferences. In this study, we have proposed a novel fluorescence resonance energy transfer (FRET)-based strategy for ultrasensitive monitoring intracellular telomerase activity with an amplification-free procedure. DNA nanoprobes constructed using a DNA tetrahedron and Flare DNA are designed to circumvent the problem of false-positive interference. Generally, telomerase catalyzes DNA extension and changes the configuration of the DNA nanoprobe, which finally leads to the increase of the distance between two labelled fluorophores. Based on the notably decreased FRET efficiency, a simple ratiometric sensor is established for the detection of telomerase activity at the single-cell level, which is much suitable for clinical applications. In addition, this method can be further applied in the detection of telomerase inhibitors, which is important for anti-cancer drug discovery.

A label-free electrochemical biosensor for ultra-sensitively detecting telomerase activity based on the enhanced catalytic currents of acetaminophen catalyzed by Au nanorods

An electrochemical biosensor was designed for the determination of telomerase activity using an enzyme-free, PCR-free, and convenient electrochemical strategy. In this work, the electrochemical biosensor was constructed through the functionalization of Au nanorods with a carboxylic group (AuNRs-3) and subsequent immobilization with capture DNA (cDNA) for sensing telomerase activity. Upon telomerase triggered extension, the telomerase activity is related to the amount of the adsorbed electrocatalyst, leading to the different electrochemical signals for readout. Integrating with the efficient electrocatalysis of AuNRs-3-cDNA towards oxidation of acetaminophen, the prepared biosensor exhibits a wide dynamic correlation of telomerase activity from 1 × 102 to 1.04 × 107 HeLa cells mL−1 with a sensitivity of 2.68 HeLa cell mL−1 and the limit of detection was calculated to be 52.81 HeLa cells mL−1 under the optimal experimental conditions. Furthermore, the application of this electrochemical biosensor would provide the great potential for analysis of telomerase activity, revealing a powerful platform for early diagnosis of cancers.

Effects of rapamycin on the mechanistic target of rapamycin (mTOR) pathway and telomerase in breast cancer cells

The mTOR pathway and the enzyme telomerase are two key players commonly upregulated in cancers. They render survival and proliferative advantage to cancer cells, and are regarded as attractive anticancer targets. Rapamycin, a macrolide antibiotic and mTOR inhibitor, has recently also been implicated in telomerase inhibition and telomere attrition, although the mechanisms remain poorly understood. Using breast cancer cells (MCF-7 and MDA-MB-231) wherein telomerase activity and mTOR pathway are concurrently overexpressed, this study sought to unravel novel mechanisms by which rapamycin may affect these pathways. Short term treatment with an acute dose of rapamycin inhibited the mTOR pathway and telomerase activity and induced G1 arrest. This arrest was independent of cyclin D1 and p21 levels and was not mediated by DNA damage in both cell types. While long term treatment with a clinically relevant dose of rapamycin resulted in compromised population doubling capacity and mTOR pathway inhibition, there was no effect on telomere functionality and telomerase activity as evidenced by our assessments of hTERT protein levels, in vitro telomerase activity, telomere length and telomere FISH analyses. We also found that sustained rapamycin treatment leading to Akt activation may play a role in resistance in the more invasive MDA-MB-231 cells. In summary, rapamycin specifically inhibits the activation of mTOR pathway. Moreover, we show for the first time that while acute short-term treatment with rapamycin induces telomerase inhibition, it does not affect telomerase activity nor does it inflict telomere dysfunction in breast cancer cells upon chronic long-term treatment with a clinically relevant dose. These findings may be useful while designing combinatorial treatment strategies with rapamycin inhibition in the clinic.

Telomerase activity in pancreatic juice differentiates pancreatic cancer from chronic pancreatitis: A meta-analysis.

To evaluate the usefulness of genetic markers in pancreatic juice (PJ), and the combination of these markers with telomerase activity in the differential diagnosis of pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis. We conducted a meta-analysis for the diagnostic utility of the four major altered genes in PDAC (KRAS, CDKN2A/p16, TP53, and SMAD4/DPC4), telomerase activity, and a combination assay using PJ samples. A literature search was conducted in MEDLINE, Cochrane Library, and Web of Science. Data were pooled and presented as diagnostic sensitivity and specificity with 95% confidence intervals (CIs). Thirty-nine studies fulfilled the inclusion criteria. Pooled estimates of KRAS analysis were as follows: sensitivity was 0.67 (95% CI, 0.63-0.71) and specificity, 0.82 (95% CI, 0.79-0.85). For telomerase activity analysis, sensitivity was 0.82 (95% CI, 0.76-0.87) and specificity, 0.96 (95% CI, 0.90-0.99). The other three tumor suppressors demonstrated low sensitivity. The data did not suggest any publication bias. A combined analysis of KRAS and telomerase activity showed a higher diagnostic sensitivity (0.94; 95% CI, 0.83-0.99) than KRAS alone. A combined analysis of telomerase activity and cytology revealed more reliable diagnostic accuracy than telomerase activity alone, with high sensitivity (0.88; 95% CI, 0.74-0.96) and specificity (1.00; 95% CI, 0.91-1.00). The most reliable marker in PJ samples for diagnosis of PDAC was telomerase activity. Telomerase activity can play a central role in diagnostic analysis using PJ samples, and can increase diagnostic accuracy when combined with KRAS mutations or cytological examination.

Activity of telomerase and telomeric length in Apis mellifera.

Telomerase is an enzyme that adds repeats of DNA sequences to the ends of chromosomes, thereby preventing their shortening. Telomerase activity is associated with proliferative status of cells, organismal development, and aging. We report an analysis of telomerase activity and telomere length in the honeybee, Apis mellifera. Telomerase activity was found to be regulated in a development and caste-specific manner. During the development of somatic tissues of larval drones and workers, telomerase activity declined to 10% of its level in embryos and remained low during pupal and adult stages but was upregulated in testes of late pupae, where it reached 70% of the embryo level. Upregulation of telomerase activity was observed in the ovaries of late pupal queens, reaching 160% of the level in embryos. Compared to workers and drones, queens displayed higher levels of telomerase activity. In the third larval instar of queens, telomerase activity reached the embryo level, and an enormous increase was observed in adult brains of queens, showing a 70-fold increase compared to a brain of an adult worker. Southern hybridization of terminal TTAGG fragments revealed a high variability of telomeric length between different individuals, although the same pattern of hybridization signals was observed in different tissues of each individual.

Abstract 539: Usefulness of genetic markers in pancreatic juice for the diagnosis of pancreatic cancer distinguishing from chronic inflammation: A meta-analysis

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) shows frequent genomic alterations in the 4 major genes, KRAS, CDKN2A/p16, TP53, and SMAD4/DPC4. Furthermore, a recent high-sensitivity mutational analysis discovered KRAS mutations in over 90% of pancreatic intraepithelial neoplasms. The genomic alterations of those genes in the pancreatic juice (PJ) have been studied for the early detection of PDAC, but the results were heterogeneous and its application was limited mainly because of the low diagnostic specificity; KRAS gene mutations have been also detected in chronic pancreatitis (CP). Therefore, for the diagnosis of early pancreatic cancer distinguishing from CP using PJ samples, more sensitive and specific method should be developed. Objective: To evaluate the diagnostic accuracy of the genetic or molecular markers in PJ samples distinguishing PDAC from CP, which is associated with a risk of pancreatic cancer development. Methods: We conducted a quantitative meta-analysis of the diagnostic studies using PJ samples, and validated the diagnostic utility of the 4 major genes, the telomerase activity, and combination assays. A systematic search of all relevant literature was performed in MEDLINE, Cochrane Database, and Web of Science. Quality assessment was conducted by adopting the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) checklist. Data are presented as forest plots and hierarchical summary receiver operating characteristic curve analysis was used to summarize the overall test performance. Results: Thirty-nine studies fulfilled the inclusion criteria, and data were pooled for the analysis. Pooled estimates for the KRAS analysis were as follows: sensitivity 0.67 (95% CI: 0.63-0.71); specificity 0.82 (95% CI: 0.79-0.85); diagnostic odds ratio (DOR) 9.24 (95% CI: 6.00-14.23). Pooled estimates for the telomerase activity analysis were: sensitivity 0.82 (95% CI: 0.76-0.87); specificity 0.96 (95% CI: 0.90-0.99); DOR 54.38 (95% CI: 21.80-135.66). All 3 tumor suppressor genes, TP53, CDKN2A/p16, and SMAD4/DPC4, demonstrated low sensitivity. The combined analysis of KRAS with telomerase activity revealed higher diagnostic sensitivity (0.94, 95% CI: 0.83-0.99) than KRAS alone. The combined assay of telomerase activity with cytology revealed more reliable diagnosis of PDAC than telomerase activity alone with respect to sensitivity (0.88, 95% CI: 0.74-0.96) and specificity (1.00, 95% CI: 0.91-1.00). Conclusion: The most reliable marker for the diagnosis of PDAC in PJ samples was telomerase activity. The assessment by each 3 major tumor suppressor was not unsatisfactory. Telomerase activity can play a central role in diagnostic analysis using PJ samples, and combination assays with KRAS mutations or cytological examination would increase diagnostic accuracy. Citation Format: Tatsuo Hata, Masaharu Ishida, Fuyuhiko Motoi, Takeshi Naitoh, Yu Katayose, Shinichi Egawa, Michiaki Unno. Usefulness of genetic markers in pancreatic juice for the diagnosis of pancreatic cancer distinguishing from chronic inflammation: A meta-analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 539. doi:10.1158/1538-7445.AM2015-539