A facile deoxyuridine/biotin-modified molecular beacon for simultaneous detection of proteins and nucleic acids via a label-free and background-eliminated fluorescence assay.

Simultaneous detection of different types of cancer biomarkers (nucleic acids and proteins) could facilitate early diagnosis of cancer and clinical treatment. Herein, a simultaneous detection platform of proteins and nucleic acids has been developed using a single substrate probe combining a label-free and background-eliminated fluorescence assay. Telomerase and telomerase RNA (TR) were chosen as the models. The molecular beacon (dU-BIO-HP) that contains deoxyuridine/biotin in its side arm, a TR recognition sequence in the loop and a telomerase substrate primer at the stem end was ingeniously designed. In the presence of telomerase, the stem of dU-BIO-HP is elongated by the addition of telomere repeats complementary to the assistant DNA. Furthermore, the formed dsDNA performed as engaging primers to initiate a SDA reaction, generating abundant G-quadruplex monomers. Similarly, on TR, the hybridization between TR and dU-BIO-HP can open its stem, triggering another SDA reaction, producing abundant short ssDNAs. With the G-quadruplex binding with ZnPPIX and ssDNA binding with SG for specific fluorescence responses, the label-free multiple detection can be achieved. In our strategy, the deoxyuridine of dU-BIO-HP acts as a barrier to block the DNA extension due to its strong inhibitory effects on DNA polymerase activity and to make sure that the two SDA reactions occurred independently. The biotin of dU-BIO-HP enables the reduction of the background from the binding between SG, ZnPPIX and dU-BIO-HP through streptavidin-biotin interaction. This method showed an excellent sensitivity with telomerase and TR detection limit of 2.18 HeLa cells per mL and 0.16 × 10-12 M, respectively. Furthermore, the telomerase and TR in different cell lines have been evaluated as powerful tools for biomedical research and clinical diagnosis.

Insight meditation and telomere biology: The effects of intensive retreat and the moderating role of personality.

A growing body of evidence suggests that meditation training may have a range of salubrious effects, including improved telomere regulation. Telomeres and the enzyme telomerase interact with a variety of molecular components to regulate cell-cycle signaling cascades, and are implicated in pathways linking psychological stress to disease. We investigated the effects of intensive meditation practice on these biomarkers by measuring changes in telomere length (TL), telomerase activity (TA), and telomere-related gene (TRG) expression during a 1-month residential Insight meditation retreat. Multilevel analyses revealed an apparent TL increase in the retreat group, compared to a group of experienced meditators, similarly comprised in age and gender, who were not on retreat. Moreover, personality traits predicted changes in TL, such that retreat participants highest in neuroticism and lowest in agreeableness demonstrated the greatest increases in TL. Changes observed in TRGs further suggest retreat-related improvements in telomere maintenance, including increases in Gar1 and HnRNPA1, which encode proteins that bind telomerase RNA and telomeric DNA. Although no group-level changes were observed in TA, retreat participants' TA levels at post-assessment were inversely related to several indices of retreat engagement and prior meditation experience. Neuroticism also predicted variation in TA across retreat. These findings suggest that meditation training in a retreat setting may have positive effects on telomere regulation, which are moderated by individual differences in personality and meditation experience. (ClinicalTrials.gov #NCT03056105).

DHA-enriched fish oil upregulates cyclin-dependent kinase inhibitor 2A (P16INK) expression and downregulates telomerase activity without modulating effects of PPARγ Pro12Ala polymorphism in type 2 diabetic patients: A randomized, double-blind, placebo-controlled clinical trial.

OBJECTIVE: The present study investigated the effects of docosahexaenoic acid (DHA)-enriched fish oil supplement on telomerase activity, mRNA expression of P16INK, IL-6, and TNF-α considering Pro12Ala polymorphism in the PPARγ gene. METHODS/DESIGN: In this double-blind randomized controlled trial, 72 PPARγ Pro12Ala polymorphism genotyped type 2 diabetic patients aged 30-70 years were randomly assigned to receive 2.4 gr of DHA-enriched fish oil or a placebo for 8 weeks. Genotyping of the Pro12Ala polymorphism in the PPARγ gene was assessed using polymerase chain reaction-restriction length polymorphism (PCR-RFLP), telomerase activity in the peripheral blood mononuclear cell (PBMC) was measured using PCR-ELISA based on the telomeric repeat amplification protocol (TRAP), and changes in the mRNA expression of P16, IL-6, and TNF-α were measured using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In the DHA group, telomerase activity was decreased (p = 0.001) during the intervention. In addition, between-group comparisons showed significant differences in the changes in telomerase activity (p = 0.003) and P16 mRNA expression (p = 0.028) and non-significant differences in TNF-α and IL-6 mRNA expression. The gene*DHA interaction could not affect changes in P16, IL-6, or TNF-α mRNA expression or in telomerase activity in PBMC. DISCUSSION: Short-time DHA-enriched fish oil supplementation caused increased levels of P16 expression and a decline in telomerase activity compared with the control group without modulating the effects of Pro12Ala polymorphism on the PPARγ gene. Because of the positive correlation between P16 activity and cellular senescence, the possibility of senescence stimulation by DHA is proposed.

New horizons for diagnostics and therapeutic applications of graphene and graphene oxide.

Graphene, a one-atom-thick two-dimensional (2D) layer of sp(2) -bonded carbon, has received worldwide attention owing to its extraordinary physical and chemical properties. Recently, great efforts have been devoted to explore potential applications of graphene and its oxide in life science, especially in disease-related diagnostics, near-Infrared (NIR) phototherapy and imaging. Here we will introduce recent advances and new horizons in this area, and focus on the rising progress on NIR photothermal therapy for cancer and Alzheimer's disease (AD), human telomerase detection, stem cell proliferation and differentiation on graphene substrate, diagnosis of cancer cell and related biomarkers, drug/nucleotide/peptide delivery and cell imaging, which have not been comprehensively reviewed. We hope to provide an outlook to the applications of graphene and its oxide, especially on the new horizons in this field, and inspire broader interests across various disciplines.

Label-free highly sensitive detection of telomerase activity in cancer cell by chemiluminescence imaging.

We have developed a new methodology for label-free highly sensitive telomerase activity assay using chemiluminescence imaging. This method can detect the telomerase activity from as little as 10 cultured cancer cells without PCR. Furthermore, telomerase inhibition is shown, demonstrating the potential for screening of telomerase inhibitors as anticancer drug agents.

Regulation of circulating progenitor cells in left ventricular dysfunction.

BACKGROUND: Reductions in numbers of circulating progenitor cells (CD34+ cell subsets) have been demonstrated in patients at risk for, or in the presence of, cardiovascular disease. The mediators of these reductions remain undefined. To determine whether neurohumoral factors might regulate circulating CD34+ cell subsets in vivo, we studied complementary canine models of left ventricular (LV) dysfunction. METHODS AND RESULTS: A pacing model of severe LV dysfunction and a hypertensive renal wrap model in which dogs were randomized to receive deoxycorticosterone acetate (DOCA) were studied. Circulating CD34+ cell subsets including hematopoietic precursor cells (HPCs: CD34+/CD45(dim)/VEGFR2-) and endothelial progenitor cells (EPCs: CD34+/CD45-/VEGFR2+) were quantified. Additionally, the effect of mineralocorticoid excess on circulating progenitor cells in normal dogs was studied. The majority of circulating CD34+ cells expressed CD45dimly and did not express VEGFR2, consistent with an HPC phenotype. HPCs were decreased in response to pacing, and this decrease correlated with plasma aldosterone levels (Spearman rank correlation=-0.67, P=0.03). In the hypertensive renal wrap model, administration of DOCA resulted in decreased HPCs. No changes were seen in EPCs in either model. Normal dogs treated with DOCA exhibited a decrease in HPCs in peripheral blood but not bone marrow associated with decreased telomerase activity. CONCLUSIONS: This is the first study to demonstrate that mineralocorticoid excess, either endogenous or exogenous, results in reduction in HPCs. These data suggest that mineralocorticoids may induce accelerated senescence of progenitor cells, leading to their reduced survival and decline in numbers.

Effects of tea polyphenols on telomerase activity of a tongue cancer cell line: a preliminary study.

The aim of this study was to determine, at the mRNA and protein levels, whether tea polyphenols (TPs) affect the expression of the human telomerase reverse transcriptase (hTERT) gene in the Tca8113 cancerous cell line. The expression of this gene was determined at the mRNA level by reverse transcription and polymerase chain reaction and at the protein level by Western blotting. The semi-quantitative scores of hTERT mRNA expression were analyzed by one-way analysis of variance. After 72 h of exposure to TPs, the mean (+/-SD) scores of hTERT mRNA expression in TP 0.1g/l, TP 0.05 g/l and a control group were 0.32+/-0.05, 0.41+/-0.04 and 0.72+/-0.05, respectively (P<0.05). The Western blot assay showed that TPs also decreased the expression of hTERT at the protein level. These results indicate that TPs reduce hTERT activity in the human Tca8113 cell line in a time- and dose-dependent manner, disabling telomerase activity and thereby terminating unlimited cancer cell proliferation. These findings suggest a mechanism behind TP's anticancer activity.

Amplified detection of telomerase activity using electrochemical and quartz crystal microbalance measurements.

Telomerase is considered as an important biomarker for cancer cells. Two different methods for the amplified electrochemical and microgravimetric quartz-crystal-microbalance detection of telomerase activity originating from HeLa cancer cells are described. One method involves the telomerization of a primer (1) linked to the electrode, in the presence of telomerase from HeLa cell extract and dNTP, followed by the hybridization of a biotin-labeled nucleic acid (2) that is complementary to the telomere repeat units. The subsequent binding of an avidin-alkaline phosphatase conjugate (3) that catalyzes the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (4) results in the precipitation of the insoluble product (5) on the electrode. The second method involves the telomerization of the primer (1) associated with the electrode, in the presence of the telomerase-containing HeLa cell extract and the dNTP nucleotide mixture that includes biotin-labeled dUTP. The telomerization leads to the labeling of the telomeres with biotin labels. The association of the avidin-alkaline phosphatase conjugate (3) to the biotin labels results in the biocatalyzed transformation of (4) to (5) and the formation of a precipitate on the electrode or the Au-quartz crystal. As numerous precipitate molecules are formed as a result of the formation of a single telomere, the methods represent routes for the amplified detection of telomerase activity. The formation of the precipitate on the respective transducers is probed by following the changes in the electrode resistance using chronopotentiometry, or by following the frequency changes of the piezoelectric quartz crystals. The amount of precipitate generated on the electrodes is controlled by the concentration of the HeLa cancer cells. The methods enable the detection of telomerase activity that is extracted from 1000 HeLa cancer cells.

[Expressions of hTERT, p53, c-erbB-2 and Bcl-2 in colonic adenocarcinoma mouse models with liver metastases: effects of classical traditional Chinese medicine prescriptions for promoting circulation and removing blood stasis].

OBJECTIVE: To observe the effects of classical traditional Chinese medicine prescriptions that function to promote blood circulation and removing blood stasis on the metastatic behavior of malignant tumors, and explore the possible mechanisms of such effects. METHODS: BALB/c mouse models of colonic adenocarcinoma with liver metastases were established by intrasplenic injection of colonic adenocarcinoma cells (CT26) to receive treatment with the three representative classical circulation-promoting and blood stasis-removing prescriptions of traditional Chinese medicine, namely Guizhifuling pills, Didang Tang and Danshenyin, respectively. The expressions of human telomerase reverse transcriptase (hTERT), p53, c-erbB-2 and Bcl-2 were detected in the mouse models with different treatments and in normal control mice. RESULTS: Tumor metastases of various degrees were observed in the mice after inoculation of the tumor cells. The expressions of hTERT, p53, c-erbB-2 and Bcl-2 in the models receiving treatment with either Didang Tang or Guizhifuling pills differed significantly from those of the untreated model group (P<0.05), whereas between Danshenyin group and the model group, only the expression of hTERT showed significant difference. CONCLUSION: Didang Tang and Guizhifuling pills can reduce the expressions of hTERT, p53, c- erbB-2 and Bcl-2, while Danshenyin can only reduce the expression of hTERT without similar effects on the other 3 oncogenes. Such inhibitory effects of the prescriptions on hTERT and the oncogenes might explain their actions to inhibit malignant tumor metastasis, which can be related to performance of the prescriptions in promoting circulation by removing blood stasis.

Association of cellular apoptosis with anti-tumor effects of the Chinese herbal complex in endocrine-resistant cancer cell line.

We previously reported the special herbal complex (Hoelen, Angelicae radix, Scutellariae radix and Glycyrrhizae radix) suppressed telomerase activity in chemo-endocrine-resistant cancer cell lines. The present study attempted to determine whether the above herbal complex induces apoptosis in endocrine-resistant AN3CA and adriamycin-resistant MCF7/ADR carcinoma cells. Exposure to the herbal complex decreased cell viability in a time- and dose-dependent manner in the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The agent induced cellular apoptosis was determined by DNA fragmentation and a nuclear staining assay. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed the decreased expression of apoptosis-related genes, bcl-2, c-myc and human telomerase catalytic subunit (hTERT). A decreased protein level of bcl-2 and c-myc was also determined by Western blot analysis. The data imply that the decreased expression of the genes via suppressing telomerase activity is involved in cellular apoptosis in endocrine-resistant AN3CA cells. Thus, it is suggested that the special herbal complex may be a promising candidate for the treatment of endocrine-resistant gynecologic carcinomas.

[Preoperative diagnostic laparoscopy with local anesthesia and lavage telomerase activity for advanced gastric cancer].

A pretherapeutic staging system to design operative or neoadjuvant treatments in gastric cancer is needed. In this study, a simple staging system based on diagnostic laparoscopic findings under local anesthesia to define a treatment was developed. This study was conducted with 68 patients with advanced gastric cancer. All the patients had been diagnosed as more than T3, or were suspected to have peritoneal seeding. Preoperative diagnostic laparoscopy under local anesthesia was performed in the operation room. After insertion of the trocars, the abdominal cavity was inspected, and biopsy and/or abdominal lavage sampling was performed. Patients were allocated into three stages based on laparoscopic and histopathologic findings. Twenty-eight out of 68 patients were diagnosed as P1, 34 patients as P0CY0 and 6 as P0CY1. Based on these results, 57 patients underwent operation. The accuracy rate of diagnosis was 95% in the P category, and 93% in the CY category. Lavage telomerase activity was examined on 10 patients with P0CY0 gastric cancer. Two out of 10 were positive for this activity, and both of them had died due to peritoneal recurrence within a year after operation. In conclusion, the proposed diagnostic laparoscopic staging system is a simple and reproducible method for selection of a suitable therapy. It is considered that diagnostic laparoscopy under local anesthesia is a useful preoperative examination in cases of advanced gastric cancer. Moreover lavage telomerase activity may be a more sensitive predictor of peritoneal recurrence than lavage cytology.

The use of telomerase activity for the detection of prostatic cancer cells after prostatic massage.

PURPOSE: Prostate cancer is the most commonly diagnosed cancer in the United States. The diagnosis or followup of prostate cancer in men older than 50 years is based on digital rectal examination, measurement of the free-to-total prostatic specific antigen ratio and transrectal ultrasound assisted needle biopsy of the prostate. We developed and evaluated a noninvasive method for diagnosing prostate cancer based on the measurement of telomerase activity after prostatic massage in fresh voided urine or after urethral washing. MATERIALS AND METHODS: We obtained 36 specimens of cells after prostatic massage in the fresh voided urine of 16 patients who subsequently underwent radical prostatectomy and after urethral washing in 20 who underwent prostate needle biopsies. Ethylenediaminetetraacetic acid was immediately added to the collected urine or washing to a final concentration of 20 mM. After protein extraction by CHAPS buffer each specimen was tested for telomerase activity in a 2-step modified telomeric repeat amplification protocol assay. The 2 prostate cancer cell lines PC-3 and LNCaP with high telomerase activity were used as a positive control. RESULTS: Telomerase activity was detected in 14 of 24 samples with known prostate cancer (sensitivity 58%). In contrast, no telomerase activity was found in the 12 cases without histological evidence of prostate tumor (specificity 100%). Eight of 9 poorly differentiated cancers expressed telomerase activity (89%), while only 6 of 15 well and moderately differentiated cancers showed telomerase activity (40%). CONCLUSIONS: Our data illustrate that telomerase activity may be detected in voided urine or washing after prostatic massage in patients with prostate cancer. Sensitivity was higher for poorly differentiated tumors. This approach is not currently available for detecting prostate cancer in clinical practice. However, these results are promising and further studies are ongoing.

Herbal complex suppresses telomerase activity in chemo-endocrine resistant cancer cell lines.

A herbal complex consisting of Hoelen, Angelicae radix, Scutellariae radix and Glycyrrhizae radix suppressed cell viability and telomerase activity in hormone-refractory and chemo-resistant cancer cell lines, namely poorly differentiated uterine endometrial cancer cell line AN3 CA, adriamycin-resistant breast cancer cell line MCF7/ADR and cisplatin-resistant ovarian cancer cell line A2780. Furthermore, the herbal complex suppressed the expression of the full length of human telomerase reverse transcriptase (hTERT), which is related to telomerase activity. This indicates that the herbal complex can suppress the tumor growth of chemoendocrine resistant cancers, at least in part via suppression of telomerase activity associated with down-regulated hTERT.

Improvement in detecting telomerase activity using silica-based resin treatment: An experience of urine in bladder carcinoma.

To improve the telomeric repeat amplification protocol (TRAP) assay to detect telomerase activity using a small amount of sample, we used a resin-column to purify and to concentrate the TS extension DNA sequence. We used 14 samples of naturally voided urine (10 ml) from patients with bladder carcinoma and 9 urine samples from patients with non-malignant urological neoplasias. We used ethylenediamine tetraacetic acid (EDTA) to stabilize telomerase activity and resin treatment to concentrate TS-extended DNA and to exclude PCR inhibitor(s), and then performed extract-based fluorescence TRAP to detect telomerase activity. None of the urinary samples without resin-column treatment had detectable telomerase activity, whereas, in resin-column treated samples, 4/9 (44%) urine samples without EDTA and 9/14 (64%) with EDTA treatment had detectable telomerase activity. A combination of EDTA treatment and resin-column thus may be available to detect telomerase activity using a relatively small amount of secretion fluids, including exfoliated urinary cells.