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In-situ Synthesized Silver Nanoclusters for Tracking the Role of Telomerase Activity in the Differentiation of Mesenchymal Stem Cells to Neural Stem Cells Fangyuan Dong, Enduo Feng, Tingting Zheng, and Yang Tian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16949 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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ACS Applied Materials & Interfaces

In-situ Synthesized Silver Nanoclusters for Tracking the Role of Telomerase Activity in the Differentiation of Mesenchymal Stem Cells to Neural Stem Cells Fangyuan Dong, Enduo, Feng, Tingting Zheng,* Yang Tian*

Shanghai Key Laboratory of Green Chemistry and Chemical Processes, Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Dongchuan Road 500, Shanghai 200241, China

*To whom correspondence should be addressed: [email protected] (T. Zheng)

[email protected] (Y. Tian)

ACS Paragon Plus Environment

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KEYWORDS Stem cells; Silver nanoclusters; In-situ synthesis; Telomerase activity; Fluorescence

ABSTRACT Human mesenchymal stem cells (hMSCs) have potential use in cell replacement therapy for central nervous system disorders. However, the factors impacted the differentiation process are unclear at the present stage, because the powerful analytical method is the bottleneck. Herein, a novel strategy was developed for self-imaging and biosensing of telomerase activity in stem cells, using in-situ bio-synthesized silver nanoclusters (AgNCs) full of C bases. The present AgNCs possesses synthetic convenience, long-time stability, and cytocompatibility. The weak fluorescence of this AgNCs quickly turned on as approaching to telomerase because of the strong interaction between C bases on AgNCs and G bases in telomerase, resulting in a telomerasedependent fluorescent signals. The developed method demonstrated high sensitivity and selectivity, broad dynamic linear range with low detection limit. Using this powerful tool, it was first discovered that telomerase activity plays the important roles in the proliferation of hMSCs and neural stem cells (NSCs) as well as during the differentiation processes from hMSCs to NSCs.


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INTRODUCTION Neural stem cells (NSC) have been a promising candidate for transplantation therapies of neurodegenerative diseases.1 However, NSC transplantation is hindered by the toxicity of immunosuppressive therapies and the limited number of donors that might be required by allogeneic transplantation.2 If NSCs could be generated from clinically accessible sources, such as the human mesenchymal cells (hMSCs), these limitations could be avoided.3 However, the factors impacted differentiation from hMSCs to NSCs are unclear at the present stage. Telomerase, a ribonucleoprotein reverse transcriptase, catalyzes the addition of TTAGGG repeats to telomeric DNA to protect telomeres from erosion during the hMSC differential process.4,5 Telomerase activity has been reported to be important in maintenance of cell proliferation.6 but the role of telomerase activity plays in proliferation of stem cells and differentiation processes from hMSCs to NSCs is still unknown, because the powerful analytical method actually is the bottleneck. Therefore, it is of great significance to open up a reliable analytical approach for in-situ monitoring of telomerase activity in proliferation and differentiation of stem cells and nerve disease therapy. A large amount of efforts have been paid on development of analytical strategies for assaying telomerase activity in the past 30 years. One of the most favorite approaches is polymerase chain reaction (PCR)-based classic telomeric repeat amplification protocol (TRAP).7 Unfortunately it is environmentally unfriendly by using radioactive

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P in the processes. Several other elegant

protocols, such as electrochemiluminescence (ECL),8 surface plasmon resonance (SPR),9 electrochemical detection,10 enzyme-linked immunosorbent assay (ELISA),11 have also been designed and employed in telomerase activity detection. With the development of fluorescence microscopic techniques, fluorescence imaging and biosensing have become powerful tools for


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Scheme 1 Schematic illustration for the biosynthesis of AgNCs for in-situ imaging and biosensing of telomerase activity. study of cellular processes.12-14 Various functionalized nanoclusters with low cytotoxicity and long-term stability has been developed as fluorescent probes in imaging of telomerase activity.15,16 Recently, increasing attention has been channeled toward biosynthesis of nanoclusters using organisms.17,18 The synthesized processes take the advantages of increasing biocompatibility, reducing the nonspecific binding, and eliminating aggregation of nanoparticles. Herein, a novel strategy was designed for in-situ biosynthesis of oligonucleotide-templated fluorescent silver nanoclusters (AgNCs), which was further developed for real-time determination of telomerase activity with high sensitivity and selectivity. Using this powerful


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tool, it was discovered that telomerase activity telomerase activity plays an important role in the proliferation of hMSCs and NSCs, as well as in the differentiation processes from hMSCs to NSCs. Firstly, AgNCs were synthesized using endogenous glutathione (GSH) in hMSCs following by treatment with silver nitrate (AgNO3) as nanocluster precursors and oligonucleotide of cytosine (C) bases as templates. The bio-synthesized AgNCs show weak fluorescence, which related to the encapsulating DNA sequences,19 but possess unique characteristics such as uniform size distribution, low cytotoxicity, long-term photostability. Interestingly, the weak fluorescence of the bio-synthesized AgNCs full of C bases turned into bright red-emission when the nanoclusters approached to guanine (G) base-rich stem elongation product (TTAGGG)n triggered by telomerase activity, as illustrated in Scheme 1, because of the specific recognition ability between G and C bases. Thus, this property provided a reliable platform to selectively detect telomerase activity through a telomerase activity-dependent fluorescence signal. This “turn-on” strategy established a simple approach for real-time imaging and biosensing of intracellular telomerase activity through one-step incubation procedure with high sensitivity as well as low detection limit. The remarkable capability for analysis of AgNCs, combined with long-term photostability and excellent biocompatibility of the biosynthesized AgNCs, provided an in-situ platform for real-time monitoring of telomerase activity during the proliferation and differentiation process of stem cells. It was found that telomerase activity plays an important role in the proliferation of hMSCs and NSCs and in the differentiation processes from hMSCs to NSCs. To the best of our knowledge, it is the first report to explore the role of telomerase activity plays in the proliferation and differentiation processes of stem cells.


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Figure 1. a) Intracellular GSH level in hMSCs during in-situ biosynthesis of c-AgNCs. Error bars represent one standard deviation for three independent measurements (S.D., n=3). b) TEM images of AgNCs located in hMSCs. c) High-resolution TEM image and d) hydrodynamic size distribution of the biosynthesized AgNCs. e) AFM image and f) XPS characterization of AgNCs. RESULTS AND DISCUSSION AgNCs were first synthesized using GSH in hMSCs following by addition of AgNO3 as precursors and C bases as template molecules. GSH is an abundant natural tripeptide presented in stem cells with high concentration (120 ± 20 nM / million cells),20 providing an inbuilt advantage for biosynthesis of AgNCs in hMSCs. GSH is a prominent reducing agent and surface stabilizer for the synthesis of AgNCs.21 During this redox reaction, GSH was oxidized to generate glutathione disulfide (GSSG) through thiol disulfide exchange as shown in Equation 1.


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2GSH + 2AgNO3

2Ag +GSSG+2HNO3

(1)

GSH concentration was tracked in the precursors-containing hMSCs during the aging process via enzymatic cycling method. Obviously, GSH was consumed during the biosynthesis of AgNCs in the first 6h and returned to its original level after 18 h of incubation (Figure 1a). The formation and localization of AgNCs in cytoplasm of hMSCs were observed from transmission electron microscopy (TEM) images (Figure 1b). The high-resolution TEM (HRTEM) image (Figure 1c) shows the clear lattice fringes of the bio-synthesized Ag NCs with an interplanar distance of 0.24 nm, corresponding to (111) plane of a Ag crystal.22 The hydrodynamic size distribution obtained from 150 clusters in a TEM image (Figure S1) indicates high monodispersity and uniform size of 2.6 ± 0.3 nm (Figure 1d). Such a narrow size distribution without agglomeration suggests that the Ag+ was reduced to AgNCs, together with generation of GSSG, resulting in the formation of AgNCs functionalized with C bases which may stabilize AgNCs well. An atomic force microscopy (AFM) image (Figure 1e) gives the topographic heights of AgNCs, which are in the range between 2.8 nm and 2.1 nm (average height is about 2.5 ± 0.2 nm). We further adopt X-ray photoelectron spectroscopy (XPS) to inspect the valence of Ag atoms in AgNCs (Figure 1f). Two peaks were clearly observed at 366.4 and 373.6 eV, which should be corresponded to the photoelectrons which excited from 3d Ag (0), indicating the formation and presence of Ag (0) NCs in hMSCs. The bio-synthesized AgNCs show weak fluorescence. However, the weak-fluorescent AgNCs remarkably “lit up” while approaching the telomerase-triggered stem elongation product (TTAGGG)n, leading to a telomerase activity-dependent fluorescence signal changes (Scheme 1). As shown in Figure 2a, with the addition of AgNO3 and GSH in hMSCs, the fluorescence intensity at 610 nm obviously increased with aging time upon excitation of 488 nm (Figure 2),


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Figure 2. a) Time course of confocal fluorescence images and bright-field images of hMSCs incubated with Ag NCs precursors for 0, 2, 6, 12, 24 and 48 h at 37 °C. All images share the same scale bar. b) Confocal fluorescence images and bright-field images of blank hMSCs (I) and hMSCs incubated with AgNO3 (II), C bases (III), AgNO3 and C bases before (IV) and after (V) 24 h postadministration and AgNO3, C bases and telomerase inhibitor (VI). All images share the same scale bar. (c) Working mechanism of c-AgNCs. revealing the developed AgNCs full of C bases were gradually generated and turned bright as approaching to telomere with rich G bases. The fluorescence intensity reached the maximum at 24 h. However, the intracellular GSH level in hMSCs decreased in the first 6h (Figure 1a) following by recovering to normal level rapidly. We could speculate that the change in fluorescence is not due to the formation of more Ag NCs, but because of the increase in emission


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Figure 3. a) UV-vis absorption (left) and fluorescence (right) spectra of AgNCs at 24 h postadministration of precursors in hMSCs. Inset: photographs of AgNCs before (left) and after (right) 24h postadministration. b) Fluorescence lifetime measurement of the biosynthesized AgNCs. c) Emission spectra of isolated AgNCs from hMSCs at different postadministration times. d) Viability (measured by MTT assay) of hMSCs incubated with AgNO3 and C bases for different times. Error bars represent one standard deviation for three independent measurements (S.D., n=3). e) Viability (measured by MTT assay) of hMSCs incubated with different concentration of AgNO3. Error bars represent one standard deviation for three independent measurements (S.D., n=3). IC50=0.078 mM.


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Table 1. the quantum yield of Ag NCs incubated with G-rich oligonucleotide for different time Time / h 0 2 6 12 24 48 QY / % 1.69 2.98 5.03. 5.96 6.41 6.39 quantum yield (QY) of Ag NCs. In control experiments, no obvious fluorescent signals were observed for blank hMSCs and hMSCs treated only with AgNO3 or cytosine-rich oligonucleotide (Figure 2b). These data confirm that the generation of AgNCs in hMSCs should be ascribed to reaction of GSH and AgNO3 according to Equation 1 inside hMSCs. Due to quantum size effect, the band of AgNCs breaks to form band gaps. Laser light induces electrons to jump between the splitting energy levels and fluorescence is observed when the excited electrons return to the ground state.23 According to the previous reports, the photoluminescence of AgNCs may be strongly related to the surface ligands.24 In our case, C base is an electron deficient group, which may block the photoluminescence pathway of AgNCs, resulting in the weak fluorescence of AgNCs. However, as AgNCs were close to G bases of telomere, which are rich in electrons and interact with C bases on AgNCs, leading to enhanced fluorescence of AgNCs (Figure 2c). To further confirm the interaction between Ag surface, C-base and G base, FT-IR spectrum was employed shown in Figure S2, Ag-DNA adduct showed several infrared bands assigned to the Ag binding to the guanine of the G and C bases at 1656, 1542, and 1480 cm-1, sugar-phosphate geometry upon Ag interaction at 1053 cm-1, and the ionic -NO3 bending vibration at 1380 cm-1 Ag-DNA adduct. 25 The biosynthesized AgNCs isolated from hMSCs after postadministration for 24 h display two UV-vis absorption peaks at 360 and 480 nm (Figure 3a). The fluorescent emission peak was located at 610 nm upon illumination of 488 nm, which agreed well with that of AgNCs in hMSCs. As shown in Table 1, the emission quantum yield (QY) was about ∼1.69%, similar to those chemically synthesized AgNCs.26 The fluorescence lifetime of the biosynthesized AgNCs


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Figure 4. a) Fluorescence spectra of the biosynthesized AgNCs in vitro in response to telomerase activity from hMSCs. b) Fluorescence spectra of AgNCs in response to telomerase activity from the 3rd of hMSCs treated with inhibitor GO with various concentrations for 12 h. c) Calibration curves for relationship between the fluorescence intensity and telomerase activity (red and blue line corresponding to a and b respectively). Error bars represent one standard deviation for three independent measure ments (S.D., n=3). d) Confocal images of different passages of hMSCs (from left to right: P1~P8) after 24 h postadministration of AgNCs precursors. Cells were pretreated without (column 1) or with (column 2) GO for 24 h before postadministration. All images in panel B share the same scale bar: 25 μm. e) Fluorescence


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spectra and intensity of hMSCs pretreated without (red) and with (blue) GO as a function of generation. Error bars represent the standard deviation of measurements on 5 samples (S.D., n=5). was measured to be 16.93 ns (Figure 3b), which was also in a good agreement with those reported in previous literatures.24 We recorded the emission spectra of the AgNCs isolated from hMSCs at 16 h, 20 h, 30 h and 36 h postadministration of the precursors to demonstrate the stability of the biosynthesized AgNCs (Figure 3c). The emission peaks and fluorescent intensities of these samples agree well with the results obtained at 24 h. The photostability of AgNCs was also examined (Figure S3): after continuous irradiation of 488 nm laser or white light for 2h, 90.2 and 93.4% of the original fluorescence intensity still retained, respectively, indicative of long-term photostability of AgNCs. Such a good photostability of Ag NCs guaranteed the reliability of this assay. During the formation of Ag NCs, the cytotoxicity of the nanocluster precursors and AgNCs formed in hMSCs was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT, Figure 3d). At 24 h postadministration, more than 90% of cells survived, suggesting that intracellular synthesis of AgNCs was safely carried out and the biosynthesized AgNCs were extremely low toxic. Consequently, this endogenous DNA as the template not only could contribute to the synthesis but also protect the cell from the toxicity of bare Ag NCs. Additionally, Figure 3e showed that the IC50 value of AgNO3 was calculated to be 0.078 mM, indicating that the amount of AgNO3 we used for incubation (0.03 mM) was non-toxic. To guarantee the whether the addition of Ag+ would influence the telomerase activity, ELISA kit was used to evaluate the influence. As shown in Figure S4, no significant change of telomerase activity was observed.


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Then, the relationship between fluorescence intensity of AgNCs full of C bases and telomerase activity in vitro were collected. As demonstrated in Figure 4a, fluorescence intensity gradually went up with increasing activity of telomerase. The calibration curve displays a linear relationship between fluorescence intensity and telomerase activity over the range of 2.78×10-14 IU to 2.37×10-11 IU (Line a in Figure 4c). For confirming the selectivity of this developed method for determination of telomerase activity in hMSCs, the cells were treated with different concentrations of dicarbonyl glyoxal (GO), a model telomerase-inhibiting drug.27 The average telomerase activity in the extract of a single hMSC was calculated through enzyme-linked immune sorbent assay (ELISA). After hMSCs (1×106 cells mL-1, 0.1 mL) were incubated with different amounts of GO for 12 h (the calculated telomerase activity in a single hMSCs were listed in Table S1), AgNO3 and oligonucleotide of C bases were added to each dish and the mixture were incubated for 24 h. The fluorescence intensity of AgNCs in live cells obtained from GO-treated hMSCs progressively decreased as the amount of GO increased (Figure 4b and S5). A calibration curve was obtained with a good linear range from 5.67×10-14 IU to 4.94×10-12 IU (Line b in Figure 4c). This curve coincided very well with Line a obtained in Figure 4c, suggesting that in situ self-imaging strategy using AgNCs shows high selectivity for determination of telomerase activity. Accordingly, our developed tool demonstrates high selectivity, high sensitivity, broad linear range and low detection limit, as well as long-term stability providing a reliable and ultrasensitive protocol for in-situ imaging and biosensing of telomerase activity, which was further used for investigating the telomerase activity in the proliferation and differentiation of stem cells.


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Figure 5. a) Cell morphologies of the 3rd generation of hMSCs during the differentiation process. All images in panel d share the same scale bar: 25 μm. b) Cell morphologies of different generations of hMSCs during the differentiation process in culture medium containing AgNCs precursors, FGF and EGF. All images share the same scale bar. c) Fluorescence microscopy images of the 3rd generation of hMSCs (column 1) and corresponding differentiated NSCs coincubated with nestin antibody and DAPI for 3 h. All images in panel c share the same scale bar: 25 μm. With the proliferation of hMSCs, fluorescence intensity of AgNCs gradually increased, and reached the maximum at the 3rd generation (Figure 4d and e). After that, the fluorescence gradually diminished as hMSCs continued to proliferate, and almost disappeared at the 6th generation. This observation indicates that the expression of telomerase was positive in the first five generations of hMSCs, and the highest telomerase activity was obtained in the 3rd generation. The results are highly consistent with those obtained using the commercial human telomerase ELISA Kit (Figure S6a). It was evident that telomerase activity plays an essential part in the proliferation processes of hMSCs.


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Figure 6. a) Bright and confocal images of different passages of NSCs (from left to right: P1~P5) containing biosynthesized AgNCs differentiated from the 3rd generation of hMSCs. All images in panel a share the same scale bar: 25 μm. b) Fluorescence intensity of NSCs as a function of generation. Error bars represent the standard deviation of measurements on 5 samples. (S.D., n=5). In order to differentiate hMSCs to NSCs, hMSCs with different generations were induced by medium containing human basic fibroblast growth factor (FGF) and human epidermal growth factor (EGF).28 All generations of the hMSCs used for neural induction displayed a fibroblastlike morphology (Figure 5a and b). After hMSCs were cultured in the differentiation medium for 8 days, hMSCs gradually aggregated and a little neurosphere-like structure began to be observed, similar to NSC-generated neurospheres in morphology. After 18 days, neurosphere-like structures were almost all observed in the first five generations of hMSCs, while the subsequent generations of hMSCs maintained the original fibroblast-like morphology. Immunocytochemical examination reveals nestin, a common NSC marker,29 was only expressed in the majority of cells in neurosphere-like structures after differentiation for the first 5 generations (Figure 5c), indicating that hMSCs were able to differentiate into NSCs till passage 5. Interestingly, as shown


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in figure 5b and S7, the final cell morphology of the 3rd generation differentiated hMSCs is most similar to that of NSCs, and the expression of nestin is the largest as compared to the NSCs differentiated from other generations of hMSCs. These results are corresponding to the telomerase activities of different generations of hMSCs demonstrated above. The 3rd generation of blank hMSCs without biosynthesized AgNCs were also induced by FGF and EGF as a control experiment. As shown in Figure S8, AgNCs has no effect on the differentiation timing and state of hMSCs. The telomerase activity of different generations of NSCs differentiated from the 3rd generation hMSCs were also observed using the bio-synthesized AgNCs (Figure 6 and S9). The telomerase was positive in the first four generations of differentiated NSCs, while negative in the subsequent passages, which was consistent with the result of ELISA kit (Figure S6b). The decrease of telomerase activity in NSCs is also one of the reasons that NSCs cannot be passaged for a long time.30 These results clearly indicate that telomerase activity is an important factor for proliferation of NSCs. CONCLUSIONS In summary, a novel “turn-on” analytical strategy has been developed for in-situ imaging and biosensing of telomerase activity through one-step biosynthesized AgNCs. The present AgNCs possess synthetic convenience, intracellular stability and low cytotoxicity. The weak fluorescence lights on as AgNCs full of C bases approaches to telomerase with G bases, due to the interaction of G-C bases. The developed method demonstrates high selectivity and sensitivity, broad linear range with low detection limit, providing a reliable and durable protocol for in-situ imaging and biosensing of telomerase activity. The remarkable analytical capability equipped


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with good biocompatibility and long-term stability, has opened up a new way to evaluating the important role of telomerase activity plays in live cells. It has been first discovered that telomerase plays an important part in the proliferation of hMSCs and NSCs as well as during the differentiation from hMSCs to NSCs. This study has not only demonstrated a new methodology for in-situ biosynthesis of fluorescent clusters, but also established an approach for imaging and biosensing of telomerase activity, which may be very powerful for further understanding of NSCs transplantation on spinal cord injury and other physiological and pathological processes.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details, Characterization, IR spectra, fluorescence spectra, Photostability, morphologies of hMSCs and NSC and ELISA kits (PDF) AUTHOR INFORMATION Corresponding Author *[email protected] (T. Zheng) *[email protected] (Y. Tian) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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The authors greatly appreciate the financial support from National Natural Science Foundation of China for distinguished Young Scholars (21325521) and NNSFC (21635003, 21175098 and 21605049). The Program of Shanghai Subject Chief Scientist (15XD1501600) ， Shanghai Sailing Program (16YF1402700) and China Postdoctoral Science Foundation (2017M610235) are gratefully acknowledged. REFERENCES 1. Goldberg, N. R. S.; Caesar, J.; Park, A.; Sedgh, S.; Finogenov, G.; Masliah, E.; Davis, J.; Blurton-Jones, M. Neural Stem Cells Rescue Cognitive and Motor Dysfunction in a Transgenic Model of Dementia with Lewy Bodies through a BDNF-Dependent Mechanism. Stem Cell Rep. 2015, 5, 791-804. 2. Hoornaert, C. J.; Blon, D. L.; Quarta, A.; Daans, J.; Goossens, H.; Berneman, Z.; Ponsaerts, P. Concise Review: Innate and Adaptive Immune Recognition of Allogeneic and Xenogeneic Cell Transplants in the Central Nervous System. Stem Cells Trans. Med. 2017, 6, 1434-1441. 3. Aggarwal, S.; Pittenger, M. F. Human Mesenchymal Stem Cells Modulate Allogeneic Immune Cell Responses. Blood 2005, 105, 1815-1822. 4. Zimmermann, S.; Voss, M.; Kaiser, S.; Kapp, U.; Waller, C. F.; Martens, U. M. Lack of Telomerase Activity in Human Mesenchymal Stem Cells. Leukemia 2003, 17, 1146-1149. 5. Smith, L. L.; Coller, H. A.; Roberts, J. M. Telomerase Modulates Expression of GrowthControlling Genes and Enhances Cell Proliferation. Nature Cell Bio. 2003, 5 474-479. 6. Greider, C. W. Telomerase Activity, Cell Proliferation, and Cancer. Proc. Natl. Acad. Sci. USA 1998, 95, 90-92.


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In Situ Synthesized Silver Nanoclusters for Tracking the Role of

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