Ultrasound-guided transversus abdominis plane block for postoperative analgesia in living liver donors: A prospective, randomized, double-blinded clinical trial.

Science.gov (United States)

Kıtlık, Arzu; Erdogan, Mehmet Ali; Ozgul, Ulku; Aydogan, Mustafa Said; Ucar, Muharrem; Toprak, Huseyin Ilksen; Colak, Cemil; Durmus, Mahmut

2017-02-01

Transversus abdominis plane (TAP) block is a peripheral nerve block that reduces postoperative pain, nausea, vomiting and the need for postoperative opioids following various types of abdominal surgery. The primary aim of the present study was to evaluate the effects of TAP block on postoperative analgesia and opioid consumption in living liver donors in whom a right "J" abdominal incision was used. This prospective, double-blinded, randomized controlled study was conducted with 50 living liver donors, aged 18-65years, who were scheduled to undergo right hepatectomy. Patients who received ultrasonography-guided subcostal TAP block were allocated into Group 1, and patients who did not receive TAP block were allocated into Group 2. The TAP blocks were performed bilaterally at the conclusion of surgery using 1.5mg∗kg -1 bupivacaine diluted with saline to reach a total volume of 40mL. For each patient, morphine consumption, pain scores at rest and movement, sedation scores, nausea, vomiting and the need for antiemetic medication were assessed at 0, 2, 4, 6, 12 and 24h postoperatively by researchers who were blinded to the study groups. Morphine consumption was significantly lower in Group 1 than in Group 2 at the 2nd, 6th and 24th hours (Pconsumption values after 24h were 40mg and 65mg in Groups 1 and 2, respectively. The TAP block significantly reduced postoperative visual analog scale pain scores both at rest and during movement at 0, 2, 4, 6, and 24h postoperatively (Pconsumption and contributed to analgesia in living liver donors who underwent upper abdominal wall incisions. Copyright © 2016 Elsevier Inc. All rights reserved.

Curcumin blocks interleukin-1 signaling in chondrosarcoma cells.

Directory of Open Access Journals (Sweden)

Thomas Kalinski

Full Text Available Interleukin (IL-1 signaling plays an important role in inflammatory processes, but also in malignant processes. The essential downstream event in IL-1 signaling is the activation of nuclear factor (NF-κB, which leads to the expression of several genes that are involved in cell proliferation, invasion, angiogenesis and metastasis, among them VEGF-A. As microenvironment-derived IL-1β is required for invasion and angiogenesis in malignant tumors, also in chondrosarcomas, we investigated IL-1β-induced signal transduction and VEGF-A expression in C3842 and SW1353 chondrosarcoma cells. We additionally performed in vitro angiogenesis assays and NF-κB-related gene expression analyses. Curcumin is a substance which inhibits IL-1 signaling very early by preventing the recruitment of IL-1 receptor associated kinase (IRAK to the IL-1 receptor. We demonstrate that IL-1 signaling and VEGF-A expression are blocked by Curcumin in chondrosarcoma cells. We further show that Curcumin blocks IL-1β-induced angiogenesis and NF-κB-related gene expression. We suppose that IL-1 blockade is an additional treatment option in chondrosarcoma, either by Curcumin, its derivatives or other IL-1 blocking agents.

Synthesis of Well-defined Amphiphilic Block Copolymers by Organotellurium-Mediated Living Radical Polymerization (TERP).

Science.gov (United States)

Kumar, Santosh; Changez, Mohammad; Murthy, C N; Yamago, Shigeru; Lee, Jae-Suk

2011-10-04

Low-molecular weight amphiphilic diblock copolymers, polystyrene-block-poly (2-vinylpyridine) (PS-b-P2VP), and (P2VP-b-PS) with different block ratios were synthesized for the first time via organotellurium-mediated living radical polymerization (TERP). For both the homo- and block copolymerizations, good agreement between the theoretical, and experimental molecular weights was found with nearly 100% yield in every case. The molecular weight distribution for all the samples ranged between 1.10 and 1.24, which is well below the theoretical lower limit of 1.50 for a conventional free radical polymerization. Furthermore, a very simple approach to producing highly dense arrays of titania nanoparticles (TiO2 ) is presented using a site-selective reaction of titanium tetraisopropoxide within the P2VP domains of micellar film of P2VP-b-PS in toluene through the sol-gel method. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Neurotrophic and X-ray blocks in the blastemal cell cycle

International Nuclear Information System (INIS)

Maden, M.

1979-01-01

Using microdensitometry techniques the points in the cycle where blastemal cells become blocked after X-irradiation or denervation of the regenerating amphibian limb have been identified. X-irradiation blocks cells in both G 1 and G 2 and those cells that were in S at the time of irradiation presumably proceed to G 2 . After denervation, however, cells accumulate only in G 1 and those that were in S or G 2 continue through the cycle to the next G 1 . The latter results are clearly contradictory to a recent theory proposing a G 2 neurotrophic control of blastemal cells and a solution to the contradiction is presented in the light of the recent results. (author)

Polyvalent Display of Biomolecules on Live Cells.

Science.gov (United States)

Shi, Peng; Zhao, Nan; Lai, Jinping; Coyne, James; Gaddes, Erin R; Wang, Yong

2018-06-04

Surface display of biomolecules on live cells offers new opportunities to treat human diseases and perform basic studies. Existing methods are primarily focused on monovalent functionalization, that is, the display of single biomolecules across the cell surface. Here we show that the surface of live cells can be functionalized to display polyvalent biomolecular structures through two-step reactions under physiological conditions. This polyvalent functionalization enables the cell surface to recognize the microenvironment one order of magnitude more effectively than with monovalent functionalization. Thus, polyvalent display of biomolecules on live cells holds great potential for various biological and biomedical applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

Science.gov (United States)

Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

Science.gov (United States)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Donor-Acceptor Block Copolymers: Synthesis and Solar Cell Applications

Directory of Open Access Journals (Sweden)

Kazuhiro Nakabayashi

2014-04-01

Full Text Available Fullerene derivatives have been widely used for conventional acceptor materials in organic photovoltaics (OPVs because of their high electron mobility. However, there are also considerable drawbacks for use in OPVs, such as negligible light absorption in the visible-near-IR regions, less compatibility with donor polymeric materials and high cost for synthesis and purification. Therefore, the investigation of non-fullerene acceptor materials that can potentially replace fullerene derivatives in OPVs is increasingly necessary, which gives rise to the possibility of fabricating all-polymer (polymer/polymer solar cells that can deliver higher performance and that are potentially cheaper than fullerene-based OPVs. Recently, considerable attention has been paid to donor-acceptor (D-A block copolymers, because of their promising applications as fullerene alternative materials in all-polymer solar cells. However, the synthesis of D-A block copolymers is still a challenge, and therefore, the establishment of an efficient synthetic method is now essential. This review highlights the recent advances in D-A block copolymers synthesis and their applications in all-polymer solar cells.

Thermoresponsive Poly(2-Oxazoline) Molecular Brushes by Living Ionic Polymerization: Modulation of the Cloud Point by Random and Block Copolymer Pendant Chains

KAUST Repository

Zhang, Ning

2012-08-10

Molecular brushes (MBs) of poly(2-oxazoline)s were prepared by living anionic polymerization of 2-isopropenyl-2-oxazoline to form the backbone and living cationic ring-opening polymerization of 2-n-propyl-2-oxazoline and 2-methyl-2-oxazoline to form random and block copolymers. Their aqueous solutions displayed a distinct thermoresponsive behavior as a function of the side-chain composition and sequence. The cloud point (CP) of MBs with random copolymer side chains is a linear function of the hydrophilic monomer content and can be modulated in a wide range. For MBs with block copolymer side chains, it was found that the block sequence had a strong and surprising effect on the CP. While MBs with a distal hydrophobic block had a CP at 70 °C, MBs with hydrophilic outer blocks already precipitated at 32 °C. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

G2-block after irradiation of cells with different p53 status

International Nuclear Information System (INIS)

Zoelzer, Friedo; Jagetia, Ganesh; Streffer, Christian

2014-01-01

Although it is clear that functional p53 is not required for radiation-induced G 2 block, certain experimental findings suggest a role for p53 in this context. For instance, as we also confirm here, the maximum accumulation in the G 2 compartment after X-ray exposure occurs much later in p53 mutants than in wild types. It remains to be seen, however, whether this difference is due to a longer block in the G 2 phase itself. We observed the movement of BrdU-labeled cells through G 2 and M into G 1 . From an analysis of the fraction of labeled cells that entered the second posttreatment cell cycle, we were able to determine the absolute duration of the G 2 and M phases in unirradiated and irradiated cells. Our experiments with four cell lines, two melanomas and two squamous carcinomas, showed that the radiation-induced delay of transition through the G 2 and M phases did not correlate with p53 status. We conclude that looking at the accumulation of cells in the G 2 compartment alone is misleading when differences in the G 2 block are investigated and that the G 2 block itself is indeed independent of functional p53. (orig.) [de

Utility of Cytospin and Cell block Technology in Evaluation of Body Fluids and Urine Samples: A Comparative Study.

Science.gov (United States)

Qamar, Irmeen; Rehman, Suhailur; Mehdi, Ghazala; Maheshwari, Veena; Ansari, Hena A; Chauhan, Sunanda

2018-01-01

Cytologic examination of body fluids commonly involves the use of direct or sediment smears, cytocentrifuge preparations, membrane filter preparations, or cell block sections. Cytospin and cell block techniques are extremely useful in improving cell yield of thin serous effusions and urine samples, and ensure high diagnostic efficacy. We studied cytospin preparations and cell block sections prepared from 180 samples of body fluids and urine samples to compare the relative efficiency of cell retrieval, preservation of cell morphology, ease of application of special stains, and diagnostic efficacy. Samples were collected and processed to prepare cytospin smears and cell block sections. We observed that overall, cell yield and preservation of individual cell morphology were better in cytospin preparations as compared to cell blocks, while preservation of architectural pattern was better in cell block sections. The number of suspicious cases also decreased on cell block sections, with increased detection of malignancy. It was difficult to prepare cell blocks from urine samples due to low cellularity. Cytospin technology is a quick, efficient, and cost-effective method of increasing cell yield in hypocellular samples, with better preservation of cell morphology. Cell blocks are better prepared from high cellularity fluids; however, tissue architecture is better studied, with improved rate of diagnosis and decrease in ambiguous results. Numerous sections can be prepared from a small amount of material. Special stains and immunochemical stains can be easily applied to cell blocks. It also provides a source of archival material.

Interactions between semiconductor nanowires and living cells.

Science.gov (United States)

Prinz, Christelle N

2015-06-17

Semiconductor nanowires are increasingly used for biological applications and their small dimensions make them a promising tool for sensing and manipulating cells with minimal perturbation. In order to interface cells with nanowires in a controlled fashion, it is essential to understand the interactions between nanowires and living cells. The present paper reviews current progress in the understanding of these interactions, with knowledge gathered from studies where living cells were interfaced with vertical nanowire arrays. The effect of nanowires on cells is reported in terms of viability, cell-nanowire interface morphology, cell behavior, changes in gene expression as well as cellular stress markers. Unexplored issues and unanswered questions are discussed.

Effect of blocking Rac1 expression in cholangiocarcinoma QBC939 cells

Directory of Open Access Journals (Sweden)

Liu Xudong

2011-05-01

Full Text Available Cholangiocarcinomas (CCs are malignant tumors that originate from epithelial cells lining the biliary tree and gallbladder. Ras correlative C3 creotoxin substrate 1 (Rac1, a small guanosine triphosphatase, is a critical mediator of various aspects of endothelial cell functions. The objective of the present investigation was to study the effect of blocking Rac1 expression in CCs. Seventy-four extrahepatic CC (ECC specimens and matched adjacent normal mucosa were obtained from the Department of Pathology, Inner Mongolia Medicine Hospital, between 2007 and 2009. Our results showed that the expression of Rac1 was significantly higher (53.12% in tumor tissues than in normal tissues. Western blotting data indicated a significant reduction in Rac1-miRNA cell protein levels. Rac1-miRNA cell growth rate was significantly different at 24, 48, and 72 h after transfection. Flow cytometry analysis showed that Rac1-miRNA cells undergo apoptosis more effectively than control QBC939 cells. Blocking Rac1 expression by RNAi effectively inhibits the growth of CCs. miRNA silencing of the Rac1 gene suppresses proliferation and induces apoptosis of QBC939 cells. These results suggest that Rac1 may be a new gene therapy target for CC. Blocking Rac1 expression in CC cells induces apoptosis of these tumor cells and may thus represent a new therapeutic approach.

4Pi-confocal microscopy of live cells

Science.gov (United States)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery.

Science.gov (United States)

Li, He; Fan, Xinqi; Chen, Xing

2016-02-01

Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidin-desthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice.

Quantification of nanowire uptake by live cells

KAUST Repository

Margineanu, Michael B.

2015-05-01

Nanostructures fabricated by different methods have become increasingly important for various applications at the cellular level. In order to understand how these nanostructures “behave” and for studying their internalization kinetics, several attempts have been made at tagging and investigating their interaction with living cells. In this study, magnetic iron nanowires with an iron oxide layer are coated with (3-Aminopropyl)triethoxysilane (APTES), and subsequently labeled with a fluorogenic pH-dependent dye pHrodo™ Red, covalently bound to the aminosilane surface. Time-lapse live imaging of human colon carcinoma HCT 116 cells interacting with the labeled iron nanowires is performed for 24 hours. As the pHrodo™ Red conjugated nanowires are non-fluorescent outside the cells but fluoresce brightly inside, internalized nanowires are distinguished from non-internalized ones and their behavior inside the cells can be tracked for the respective time length. A machine learning-based computational framework dedicated to automatic analysis of live cell imaging data, Cell Cognition, is adapted and used to classify cells with internalized and non-internalized nanowires and subsequently determine the uptake percentage by cells at different time points. An uptake of 85 % by HCT 116 cells is observed after 24 hours incubation at NW-to-cell ratios of 200. While the approach of using pHrodo™ Red for internalization studies is not novel in the literature, this study reports for the first time the utilization of a machine-learning based time-resolved automatic analysis pipeline for quantification of nanowire uptake by cells. This pipeline has also been used for comparison studies with nickel nanowires coated with APTES and labeled with pHrodo™ Red, and another cell line derived from the cervix carcinoma, HeLa. It has thus the potential to be used for studying the interaction of different types of nanostructures with potentially any live cell types.

Long-Term Results of Stent Placement in Patients with Outflow Block After Living-Donor-Liver Transplantation

Energy Technology Data Exchange (ETDEWEB)

Fujimori, Masashi, E-mail: fujimorim@clin.medic.mie-u.ac.jp [Mie University School of Medicine, Department of Radiology (Japan); Yamakado, Koichiro, E-mail: yamakado47@gmail.com; Takaki, Haruyuki, E-mail: takaki-h@clin.medic.mie-u.ac.jp [Hyogo College of Medicine, Department of Radiology (Japan); Nakatsuka, Atsuhiro, E-mail: nakatuka@clin.medic.mie-u.ac.jp; Uraki, Junji, E-mail: junji@clin.medic.mie-u.ac.jp; Yamanaka, Takashi, E-mail: t-yama@clin.medic.mie-u.ac.jp; Hasegawa, Takaaki, E-mail: hasegawat@clin.medic.mie-u.ac.jp; Sugino, Yuichi, E-mail: ysugino23@clin.medic.mie-u.ac.jp; Nakajima, Ken, E-mail: k-nakajima@clin.medic.mie-u.ac.jp; Matsushita, Naritaka, E-mail: n-matsushita@clin.medic.mie-u.ac.jp [Mie University School of Medicine, Department of Radiology (Japan); Mizuno, Shugo, E-mail: mizunos@clin.medic.mie-u.ac.jp [Mie University School of Medicine, Hepatobiliary Pancreatic and Transplant Surgery (Japan); Sakuma, Hajime, E-mail: sakuma.mie@gmail.com [Mie University School of Medicine, Department of Radiology (Japan); Isaji, Shuji, E-mail: isaji-s@clin.medic.mie-u.ac.jp [Mie University School of Medicine, Hepatobiliary Pancreatic and Transplant Surgery (Japan)

2016-04-15

PurposeTo evaluate long-term results of stent placement retrospectively in patients with outflow block after living-donor-liver transplantation (LDLT).Materials and MethodsFor this institutional review board approved retrospective study conducted during 2002–2012, stents were placed in outflow veins in 15 patients (11.3 %, 15/133) (12 men; 3 female) in whom outflow block developed after LDLT. Their mean age was 52.3 years ± 15.3 (SD) (range, 4–69 years). Venous stenosis with a pressure gradient ≥5 mmHg (outflow block) was observed in the inferior vena cava in seven patients, hepatic vein in seven patients, and both in one patient. Technical success, change in a pressure gradient and clinical manifestations, and complications were evaluated. Overall survival of 15 patients undergoing outflow block stenting was compared with that of 116 patients without outflow block after LDLT.ResultsStents were placed across the outflow block veins without complications, lowering the pressure gradient ≤ 3 mmHg in all patients (100 %, 15/15). Clinical manifestations improved in 11 patients (73.3 %, 11/15), and all were discharged from the hospital. However, they did not improve in the other 4 patients (26.7 %, 4/15) who died in the hospital 1.0–3.7 months after stenting (mean, 2.0 ± 1.2 months). No significant difference in 5-year survival rates was found between patients with and without outflow block after LDLT (61.1 vs. 72.2 %, p = .405).ConclusionStenting is a feasible, safe, and useful therapeutic option to resolve outflow block following LDLT, providing equal survival to that of patients without outflow block.

Microencapsulation Of Living Cells

Science.gov (United States)

Chang, Manchium; Kendall, James M.; Wang, Taylor G.

1989-01-01

In experimental technique, living cells and other biological materials encapsulated within submillimeter-diameter liquid-filled spheres. Sphere material biocompatible, tough, and compliant. Semipermeable, permitting relatively small molecules to move into and out of sphere core but preventing passage of large molecules. New technique promises to make such spherical capsules at high rates and in uniform, controllable sizes. Capsules injected into patient through ordinary hypodermic needle. Promising application for technique in treatment of diabetes. Also used to encapsulate pituitary cells and thyroid hormone adrenocortical cells for treatment of other hormonal disorders, to encapsulate other secreting cells for transplantation, and to package variety of pharmaceutical products and agricultural chemicals for controlled release.

Live cell refractometry using microfluidic devices.

Science.gov (United States)

Lue, Niyom; Popescu, Gabriel; Ikeda, Takahiro; Dasari, Ramachandra R; Badizadegan, Kamran; Feld, Michael S

2006-09-15

Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.

Living cationic polymerization and polyhomologation: an ideal combination to synthesize functionalized polyethylene–polyisobutylene block copolymers

KAUST Repository

Zhang, Hefeng

2015-12-17

A series of hydroxyl-terminated polyisobutylene-b-polyethylene (PIB-b-PE-OH) copolymers were synthesized by combining living cationic polymerization and polyhomologation. Allyl-terminated PIBs, synthesized by living cationic polymerization, were hydroborated with BH3·THF to produce 3-arm boron-linked stars, PIB3B, which served as macroinitiators for the in situ polyhomologation of dimethylsulfoxonium methylide. The resulting 3-arm star block copolymers, (PIB-b-PE)3B, were oxidized/hydrolysed to afford PIB-b-PE-OH. Characterization of all intermediates and final products by high temperature gel permeation chromatography (HT-GPC) and proton nuclear magnetic resonance spectroscopy (1H NMR) revealed the well-defined character of the copolymers. The thermal properties of the copolymers were studied by differential scanning calorimetry (DSC).

SYNTHESIS OF BLOCK COPOLYMER BY INTEGRATED LIVING ANIONIC POLYMERIZATION-ATOM TRANSFER RADICAL POLYMERIZATION (ATRP)

Institute of Scientific and Technical Information of China (English)

Bing Liu; Feng Liu; Ning Luo; Sheng-kang Ying; Qing Liu

2000-01-01

Alpha-trichloroacetoxy terminated polystyrene oligomer (PS-CH2CH2OCOCCl3) and poly-(styrene-b-butadiene)oligomer [P(S-b-B)-CH2CH2OCOCCl3)] were synthesized by living anionic polymeri-zation using n-butyllithium as initiator.Then the PS-CH2CH2OCOCCl3 (PS-Cl3) or P(S-b-B)-CH2CH2O-COCCl3 (PSB-Cl3) was used as the macroinitiator in the polymerization of (meth)acrylates in the presence of CuX/bpy. AB diblock and ABC triblock copolymers were prepared by the integrated living anionic polymerization (LAP)-atom transfer radical polymerization (ATRP). The structures of the PSB-Cl3 and the P(S-b-MMA) were identified by FTIR and 1H-NMR spectrum, respectively. A new way to design block copolymers (the combination of LAP and ATRP) was developed.

Recent advances in live cell imaging of hepatoma cells

Science.gov (United States)

2014-01-01

Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

Functional living biointerfaces to direct cell-material interaction

OpenAIRE

Rodrigo Navarro, Aleixandre

2016-01-01

[EN] This thesis deals with the development of a living biointerface between synthetic substrates and living cells to engineer cell-material interactions for tissue engineering purposes. This living biointerface is made of Lactococcus lactis, a non-pathogenic lactic bacteria widely used as starter in the dairy industry and, recently, in the expression of heterologous proteins in applications such as oral vaccine delivery or membrane-bound expression of proteins. L. lactis has been engine...

G2-block after irradiation of cells with different p53 status

Energy Technology Data Exchange (ETDEWEB)

Zoelzer, Friedo [University of South Bohemia in Ceske Budejovice, Department of Radiology, Toxicology and Civil Protection, Faculty of Health and Social Studies, Ceske Budejovice (Czech Republic); University Duisburg-Essen, Institute of Medical Radiobiology, Medical Faculty, Essen (Germany); Jagetia, Ganesh [University Duisburg-Essen, Institute of Medical Radiobiology, Medical Faculty, Essen (Germany); Mizoram University, Department of Zoology, School of Life Sciences, Aizawl (India); Streffer, Christian [University Duisburg-Essen, Institute of Medical Radiobiology, Medical Faculty, Essen (Germany)

2014-11-15

Although it is clear that functional p53 is not required for radiation-induced G{sub 2} block, certain experimental findings suggest a role for p53 in this context. For instance, as we also confirm here, the maximum accumulation in the G{sub 2} compartment after X-ray exposure occurs much later in p53 mutants than in wild types. It remains to be seen, however, whether this difference is due to a longer block in the G{sub 2} phase itself. We observed the movement of BrdU-labeled cells through G{sub 2} and M into G{sub 1}. From an analysis of the fraction of labeled cells that entered the second posttreatment cell cycle, we were able to determine the absolute duration of the G{sub 2} and M phases in unirradiated and irradiated cells. Our experiments with four cell lines, two melanomas and two squamous carcinomas, showed that the radiation-induced delay of transition through the G{sub 2} and M phases did not correlate with p53 status. We conclude that looking at the accumulation of cells in the G{sub 2} compartment alone is misleading when differences in the G{sub 2} block are investigated and that the G{sub 2} block itself is indeed independent of functional p53. (orig.) [German] Obwohl klar ist, dass ein funktionelles p53-Protein fuer die Ausbildung des strahleninduzierten G{sub 2}-Blocks nicht zwingend erforderlich ist, gibt es experimentelle Befunde, die nahe legen, dass p53 in diesem Zusammenhang doch eine gewisse Rolle spielt. Zum Beispiel bestaetigen wir hier fruehere Berichte, dass die Akkumulation von Zellen im G{sub 2}-Kompartiment bei p53-Mutanten deutlich spaeter nach Bestrahlung ihr Maximum erreicht als bei p53-Wildtypen. Es bleibt jedoch zu klaeren, ob dieser Unterschied seinen Grund in einem laengeren Block der G{sub 2}-Phase selbst hat. Beobachtet wurde die Bewegung von BrdU-markierten Zellen durch G{sub 2} und M nach G{sub 1}. Aus der zeitlichen Veraenderung des Anteils markierter Zellen im G{sub 1}-Kompartiment des naechsten Zellzyklus konnte die

The contribution of cell blocks in the diagnosis of mediastinal masses and hilar adenopathy samples from echobronchoscopy.

Science.gov (United States)

Lourido-Cebreiro, Tamara; Leiro-Fernández, Virginia; Tardio-Baiges, Antoni; Botana-Rial, Maribel; Núñez-Delgado, Manuel; Álvarez-Martín, M Jesús; Fernández-Villar, Alberto

2014-07-01

Cell block material from puncture can be obtained with endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in many cases. The aim of this study was to analyse the value of additional information from cell blocks obtained with EBUS-TBNA samples from mediastinal and hilar lymph nodes and masses. Review of pathology reports with a specific diagnosis obtained from EBUS-TBNA samples of mediastinal or hilar lesions, prospectively obtained over a two-year period. The generation of cell blocks from cytology needle samples, the contribution to morphological diagnosis, and the possible use of samples for immunohistochemistry were analysed. One hundred and twenty-nine samples corresponding to 110 patients were reviewed. The diagnosis was lung cancer in 81% of cases, extrapulmonary carcinoma in 10%, sarcoidosis in 4%, lymphoma in 2.7%, and tuberculosis in 0.9%. Cell blocks could be obtained in 72% of cases. Immunohistochemistry studies on the cell blocks were significantly easier to perform than on conventional smears (52.6% vs. 14%, P<.0001). In 4cases, the cell block provided an exclusive morphological diagnosis (3sarcoidosis and one metastasis from prostatic carcinoma) and in 3carcinomas, subtype and origin could be identified. Exclusive diagnoses from the cell block were significantly more frequent in benign disease than in malignant disease (25% vs 0.9%, P=.002). Cell blocks were obtained from 72% of EBUS-TBNA diagnostic procedures. The main contributions of cell blocks to pathology examinations were the possibility of carrying out immunohistochemical staining for the better classification of neoplasms, especially extrapulmonary metastatic tumours, and the improved diagnosis of benign lesions. Copyright © 2013 SEPAR. Published by Elsevier Espana. All rights reserved.

Live cell imaging of in vitro human trophoblast syncytialization.

Science.gov (United States)

Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

2014-06-01

Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

CD47-CAR-T Cells Effectively Kill Target Cancer Cells and Block Pancreatic Tumor Growth.

Science.gov (United States)

Golubovskaya, Vita; Berahovich, Robert; Zhou, Hua; Xu, Shirley; Harto, Hizkia; Li, Le; Chao, Cheng-Chi; Mao, Mike Ming; Wu, Lijun

2017-10-21

CD47 is a glycoprotein of the immunoglobulin superfamily that is often overexpressed in different types of hematological and solid cancer tumors and plays important role in blocking phagocytosis, increased tumor survival, metastasis and angiogenesis. In the present report, we designed CAR (chimeric antigen receptor)-T cells that bind CD47 antigen. We used ScFv (single chain variable fragment) from mouse CD47 antibody to generate CD47-CAR-T cells for targeting different cancer cell lines. CD47-CAR-T cells effectively killed ovarian, pancreatic and other cancer cells and produced high level of cytokines that correlated with expression of CD47 antigen. In addition, CD47-CAR-T cells significantly blocked BxPC3 pancreatic xenograft tumor growth after intratumoral injection into NSG mice. Moreover, we humanized mouse CD47 ScFv and showed that it effectively bound CD47 antigen. The humanized CD47-CAR-T cells also specifically killed ovarian, pancreatic, and cervical cancer cell lines and produced IL-2 that correlated with expression of CD47. Thus, CD47-CAR-T cells can be used as a novel cellular therapeutic agent for treating different types of cancer.

Detecting and Tracking Nonfluorescent Nanoparticles Probes in Live Cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Gufeng; Fang, Ning

2012-01-17

Precisely imaging and tracking dynamic biological processes in live cells are crucial for both fundamental research in life sciences and biomedical applications. Nonfluorescent nanoparticles are emerging as important optical probes in live-cell imaging because of their excellent photostability, large optical cross sections, and low cytotoxicity. Here, we provide a review of recent development in optical imaging of nonfluorescent nanoparticle probes and their applications in dynamic tracking and biosensing in live cells. A brief discussion on cytotoxicity of nanoparticle probes is also provided.

Long term imaging of living brain cancer cells

Science.gov (United States)

Farias, Patricia M. A.; Galembeck, André; Milani, Raquel; Andrade, Arnaldo C. D. S.; Stingl, Andreas

2018-02-01

QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

Fine needle aspiration cytology and cell block in the diagnosis of seminoma testis

Directory of Open Access Journals (Sweden)

Abhishant Pandey

2011-01-01

Full Text Available Testicular neoplasms which show a wide variety of morphologic types, comprise a small proportion of malignancies. Early identification and treatment is essential for achieving long term survival. The cytologic findings in fine needle aspiration smears from left testicular swelling of a 49 year old male suggestive of a germ cell tumor was complimented by cell block preparation as seminoma. This was confirmed by histopathologic studies. We are presenting this case to emphasize that cell block can be used for diagnosis of testicular tumors.

Kinase Activity Studied in Living Cells Using an Immunoassay

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Bavec, Aljos?a

2014-01-01

This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

Assessing resolution in live cell structured illumination microscopy

Science.gov (United States)

Pospíšil, Jakub; Fliegel, Karel; Klíma, Miloš

2017-12-01

Structured Illumination Microscopy (SIM) is a powerful super-resolution technique, which is able to enhance the resolution of optical microscope beyond the Abbe diffraction limit. In the last decade, numerous SIM methods that achieve the resolution of 100 nm in the lateral dimension have been developed. The SIM setups with new high-speed cameras and illumination pattern generators allow rapid acquisition of the live specimen. Therefore, SIM is widely used for investigation of the live structures in molecular and live cell biology. Quantitative evaluation of resolution enhancement in a real sample is essential to describe the efficiency of super-resolution microscopy technique. However, measuring the resolution of a live cell sample is a challenging task. Based on our experimental findings, the widely used Fourier ring correlation (FRC) method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Therefore, the resolution assessing methods based on Fourier spectrum analysis are often used. We introduce a measure based on circular average power spectral density (PSDca) estimated from a single SIM image (one video frame). PSDca describes the distribution of the power of a signal with respect to its spatial frequency. Spatial resolution corresponds to the cut-off frequency in Fourier space. In order to estimate the cut-off frequency from a noisy signal, we use a spectral subtraction method for noise suppression. In the future, this resolution assessment approach might prove useful also for single-molecule localization microscopy (SMLM) live cell imaging.

Analysis of live cell images: Methods, tools and opportunities.

Science.gov (United States)

Nketia, Thomas A; Sailem, Heba; Rohde, Gustavo; Machiraju, Raghu; Rittscher, Jens

2017-02-15

Advances in optical microscopy, biosensors and cell culturing technologies have transformed live cell imaging. Thanks to these advances live cell imaging plays an increasingly important role in basic biology research as well as at all stages of drug development. Image analysis methods are needed to extract quantitative information from these vast and complex data sets. The aim of this review is to provide an overview of available image analysis methods for live cell imaging, in particular required preprocessing image segmentation, cell tracking and data visualisation methods. The potential opportunities recent advances in machine learning, especially deep learning, and computer vision provide are being discussed. This review includes overview of the different available software packages and toolkits. Copyright © 2017. Published by Elsevier Inc.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

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Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu

2016-04-01

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM

78 FR 49528 - Consolidation of Wound Care Products Containing Live Cells

Science.gov (United States)

2013-08-14

...] Consolidation of Wound Care Products Containing Live Cells AGENCY: Food and Drug Administration, HHS. ACTION... certain wound care products containing live cells from the Center for Devices and Radiological Health... CDRH and CBER. FDA believes that as more wound care products containing live cells are developed such...

Optical imaging of non-fluorescent nanoparticle probes in live cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Gufeng; Stender, Anthony S.; Sun, Wei; and Fang, Ning

2009-12-17

Precise imaging of cellular and subcellular structures and dynamic processes in live cells is crucial for fundamental research in life sciences and in medical applications. Non-fluorescent nanoparticles are an important type of optical probe used in live-cell imaging due to their photostability, large optical cross-sections, and low toxicity. Here, we provide an overview of recent developments in the optical imaging of non-fluorescent nanoparticle probes in live cells.

Induction of Live Cell Phagocytosis by a Specific Combination of Inflammatory Stimuli

Directory of Open Access Journals (Sweden)

Takamasa Ishidome

2017-08-01

Full Text Available Conditions of severe hyper-inflammation can lead to uncontrolled activation of macrophages, and the ensuing phagocytosis of live cells. However, relationships between inflammatory stimuli and uncontrolled phagocytosis of live cells by macrophages are poorly understood. To identify mediators of this process, we established phagocytosis assays of live cells by stimulating macrophages with CpG DNA, interferon-γ, and anti-interleukin-10 receptor antibody. In this model, various cell surface receptors were upregulated on macrophages, and phagocytosis of live cells was induced in a Rac1-dependent manner. Subsequent inhibition of the ICAM-1, VCAM-1, and both of these receptors abolished in vitro and in vivo phagocytosis of live T cells, myeloid cells, and B cells, respectively. Specifically, the reduction in lymphocyte numbers due to in vivo activation of macrophages was ameliorated in Icam-1-deficient mice. In addition, overexpression of ICAM-1 or VCAM-1 in non-phagocytic NIH3T3 cells led to active phagocytosis of live cells. These data indicate molecular mechanisms underlying live cell phagocytosis induced by hyper-inflammation, and this experimental model will be useful to clarify the pathophysiological mechanisms of hemophagocytosis and to indicate therapeutic targets.

Live cell imaging reveals at novel view of DNA

International Nuclear Information System (INIS)

Moritomi-Yano, Keiko; Yano, Ken-ichi

2010-01-01

Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

Information management for high content live cell imaging

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White Michael RH

2009-07-01

Full Text Available Abstract Background High content live cell imaging experiments are able to track the cellular localisation of labelled proteins in multiple live cells over a time course. Experiments using high content live cell imaging will generate multiple large datasets that are often stored in an ad-hoc manner. This hinders identification of previously gathered data that may be relevant to current analyses. Whilst solutions exist for managing image data, they are primarily concerned with storage and retrieval of the images themselves and not the data derived from the images. There is therefore a requirement for an information management solution that facilitates the indexing of experimental metadata and results of high content live cell imaging experiments. Results We have designed and implemented a data model and information management solution for the data gathered through high content live cell imaging experiments. Many of the experiments to be stored measure the translocation of fluorescently labelled proteins from cytoplasm to nucleus in individual cells. The functionality of this database has been enhanced by the addition of an algorithm that automatically annotates results of these experiments with the timings of translocations and periods of any oscillatory translocations as they are uploaded to the repository. Testing has shown the algorithm to perform well with a variety of previously unseen data. Conclusion Our repository is a fully functional example of how high throughput imaging data may be effectively indexed and managed to address the requirements of end users. By implementing the automated analysis of experimental results, we have provided a clear impetus for individuals to ensure that their data forms part of that which is stored in the repository. Although focused on imaging, the solution provided is sufficiently generic to be applied to other functional proteomics and genomics experiments. The software is available from: fhttp://code.google.com/p/livecellim/

A cell transportation solution that preserves live circulating tumor cells in patient blood samples

International Nuclear Information System (INIS)

Stefansson, Steingrimur; Adams, Daniel L.; Ershler, William B.; Le, Huyen; Ho, David H.

2016-01-01

Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90 % viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs

A cell transportation solution that preserves live circulating tumor cells in patient blood samples.

Science.gov (United States)

Stefansson, Steingrimur; Adams, Daniel L; Ershler, William B; Le, Huyen; Ho, David H

2016-05-06

Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90% viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs after

Randomized clinical trial of transversus abdominis plane block versus placebo control in live-donor nephrectomy.

Science.gov (United States)

Hosgood, Sarah A; Thiyagarajan, Umasanker M; Nicholson, Harriet F L; Jeyapalan, Inthira; Nicholson, Michael L

2012-09-15

Laparoscopic surgery reduces pain after donor nephrectomy; however, most patients still require a significant amount of postoperative parenteral opiate analgesia. Therefore, there is a need to investigate techniques that might further reduce postoperative pain. This study assessed the safety and efficacy of using a transversus abdominis plane (TAP) block in a randomized, double-blind, placebo-controlled trial. Forty-six patients were analyzed in the trial and were randomized to undergo the TAP block procedure with either bupivacaine (n=24) or saline placebo (Control n=22) injected into the muscle plane. Prefilled syringes were dispensed with the group allocation concealed to maintain blinding. After surgery, the amount of morphine, level of pain, and measures of recovery were recorded. The amount of morphine used 6 hr after surgery was significantly lower in patients receiving TAP block with bupivacaine compared with the control (presented as mean [SD], 12.4 [8.4] vs. 21.2 [14.0] mg; P=0.015). However, the total amount of morphine used was similar in both groups 45.6 [31.4] vs. 52.7 [28.8] mg; P=0.771. Patients in the bupivacaine group experienced significantly less pain on postoperative days 1 (score, 19 [15] vs. 37 [20]; P=0.003) and 2 (score, 11 [10] vs. 19 [13]; P=0.031). Recovery and postoperative hospital stay were similar in both groups. There were no complications associated with the procedure. The TAP block procedure is beneficial in reducing postoperative pain and early morphine requirements in laparoscopic live-donor nephrectomy.

PeakForce Tapping resolves individual microvilli on living cells.

Science.gov (United States)

Schillers, Hermann; Medalsy, Izhar; Hu, Shuiqing; Slade, Andrea L; Shaw, James E

2016-02-01

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip-sample interactions in time (microseconds) and controlling force in the low pico-Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM-Live Cell probe, having a short cantilever with a 17-µm-long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico-Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

Laser-Raman spectroscopy of living cells

International Nuclear Information System (INIS)

Webb, S.J.

1980-01-01

Investigations into the laser-Raman shift spectra of bacterial and mammalian cells have revealed that many Raman lines observed at 4-6 K, do not appear in the spectra of cells held at 300 K. At 300 K, Raman activity, at set frequencies, is observed only when the cells are metabolically active; however, the actual live cell spectrum, between 0 and 3400 cm -1 , has been found to alter in a specific way with time as the cells' progress through their life cycles. Lines above 300 cm -1 , from in vivo Raman active states, appear to shift to higher wave numbers whereas those below 300 cm -1 seem to shift to lower ones. The transient nature of many shift lines observed and the intensity of them when present in the spectrum indicates that, in, vivo, a metabolically induced condensation of closely related states occurs at a set time in the life of a living cell. In addition, the calculated ratio between the intensities of Stokes and anti-Stokes lines observed suggests that the metabolically induced 'collective' Raman active states are produced, in vivo, by non thermal means. It appears, therefore, that the energetics of the well established cell 'time clock' may be studied by laser-Raman spectroscopy; moreover, Raman spectroscopy may yield a new type of information regarding the physics of such biological phenomena as nutrition, virus infection and oncogenesis. (orig.)

Circulating blocking factors of lymphoid-cell cytotoxicity in x-ray-induced rat small-bowel adenocarcinoma

International Nuclear Information System (INIS)

Stevens, R.H.; Brooks, G.P.; Osborne, J.W.

1979-01-01

Circulating blocking factors capable of abrogating cell-mediated immune responses measured by in vitro lymphoid-cell cytotoxicity were identified in the sera of Holtzman outbred rats 6 to 9 months after a single exposure of only the temporarily exteriorized, hypoxic ileum and jejunum to 1700 to 2000 R of X radiation. Such factors were found to exist in the serum of every animal exposed to the ionizing radiation regardless of whether a visibly identifiable small-bowel adenocarcinoma existed or subsequently would develop. Protection of cultured x-ray-induced rat small-bowel cancer cells from destruction by tumor-sensitized lymphoid cells as measured by the release of lactoperoxidase-catalyzed radioiodinated membrane proteins from the tumor target cells was conferred by the action of the blocking factors at both effector and target cell levels. The results of this study demonstrate that exposure of only the rat small intestine to ionizing radiation leads to elaboration of circulating factors identifiable several months postirradiation which will block cell-mediated immune responses directed against cancer cells developing in the exposed tissue

Microencapsulating and Banking Living Cells for Cell-Based Medicine

Directory of Open Access Journals (Sweden)

Wujie Zhang

2011-01-01

Full Text Available A major challenge to the eventual success of the emerging cell-based medicine such as tissue engineering, regenerative medicine, and cell transplantation is the limited availability of the desired cell sources. This challenge can be addressed by cell microencapsulation to overcome the undesired immune response (i.e., to achieve immunoisolation so that non-autologous cells can be used to treat human diseases, and by cell/tissue preservation to bank living cells for wide distribution to end users so that they are readily available when needed in the future. This review summarizes the status quo of research in both cell microencapsulation and banking the microencapsulated cells. It is concluded with a brief outlook of future research directions in this important field.

AFM review study on pox viruses and living cells.

Science.gov (United States)

Ohnesorge, F M; Hörber, J K; Häberle, W; Czerny, C P; Smith, D P; Binnig, G

1997-10-01

Single living cells were studied in growth medium by atomic force microscopy at a high--down to one image frame per second--imaging rate over time periods of many hours, stably producing hundreds of consecutive scans with a lateral resolution of approximately 30-40 nm. The cell was held by a micropipette mounted onto the scanner-piezo as shown in Häberle, W., J. K. H. Hörber, and G. Binnig. 1991. Force microscopy on living cells. J. Vac. Sci. Technol. B9:1210-0000. To initiate specific processes on the cell surface the cells had been infected with pox viruses as reported earlier and, most likely, the liberation of a progeny virion by the still-living cell was observed, hence confirming and supporting earlier results (Häberle, W., J. K. H. Hörber, F. Ohnesorge, D. P. E. Smith, and G. Binnig. 1992. In situ investigations of single living cells infected by viruses. Ultramicroscopy. 42-44:1161-0000; Hörber, J. K. H., W. Häberle, F. Ohnesorge, G. Binnig, H. G. Liebich, C. P. Czerny, H. Mahnel, and A. Mayr. 1992. Investigation of living cells in the nanometer regime with the atomic force microscope. Scanning Microscopy. 6:919-930). Furthermore, the pox viruses used were characterized separately by AFM in an aqueous environment down to the molecular level. Quasi-ordered structural details were resolved on a scale of a few nm where, however, image distortions and artifacts due to multiple tip effects are probably involved--just as in very high resolution (small dark spots in the light microscope, that we believed to be the regions in the cell plasma where viruses are assembled; this is known from the literature on electron microscopy on pox-infected cells and referred to there as "virus factories" (e.g., Moss, B. 1986. Replication of pox viruses. In Fundamental Virology, B. N. Fields and D. M. Knape, editors. Raven Press, New York. 637-655). Therefore, we assume that the cells stay alive during imaging, in our experience for approximately 30-45 h p.i.).

Live cell microscopy of DNA damage response in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Pinela da Silva, Sonia Cristina; Gallina, Irene; Eckert-Boulet, Nadine Valerie

2012-01-01

live cell imaging allows for multiple cellular markers to be monitored over several hours. This chapter reviews useful fluorescent markers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for live cell imaging, time-lapse microscopy, and for induction...

Cell Penetrating Polymers Containing Guanidinium Trigger Apoptosis in Human Hepatocellular Carcinoma Cells unless Conjugated to a Targeting N-Acetyl-Galactosamine Block.

Science.gov (United States)

Tan, Zhe; Dhande, Yogesh K; Reineke, Theresa M

2017-12-20

A series of 3-guanidinopropyl methacrylamide (GPMA)-based polymeric gene delivery vehicles were developed via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers have been evaluated for their cellular internalization ability, transfection efficiency, and cytotoxicity. Two homopolymers: P(GPMA 20 ), P(GPMA 34 ), were synthesized to study the effect of guanidium polymer length on delivery efficiency and toxicity. In addition, an N-acetyl-d-galactosamine (GalNAc)-based hydrophilic block was incorporated to produce diblock polymers, which provides a neutral hydrophilic block that sterically protects plasmid-polymer complexes (polyplexes) from colloidal aggregation and aids polyplex targeting to hepatocytes via binding to asialoglycoprotein receptors (ASGPRs). Polyplexes formed with P(GPMA x ) (x = 20, 34) homopolymers were shown to be internalized via both energy-dependent and independent pathways, whereas polyplexes formed with block polymers were internalized through endocytosis. Notably, P(GPMA x ) polyplexes enter cells very efficiently but are also very toxic to human hepatocellular carcinoma (HepG2) cells and triggered cell apoptosis. In comparison, the presence of a carbohydrate block in the polymer structures reduced the cytotoxicity of the polyplex formulations and increased gene delivery efficiency with HepG2 cells. Transfection efficiency and toxicity studies were also carried out with HEK 293T (human embryonic kidney) cells for comparison. Results showed that polyplexes formed with the P(GPMA x ) homopolymers exhibit much higher transfection efficiency and lower toxicity with HEK 293T cells. The presence of the carbohydrate block did not further increase transfection efficiency in comparison to the homopolymers with HEK 293T cells, likely due to the lack of ASGPRs on the HEK 293T cell line. This study revealed that although guanidinium-based polymers have high membrane permeability, their application as plasmid

Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

Science.gov (United States)

Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A; Gratton, Enrico; Jones, Mark R

2015-05-27

DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.

Saponins from soy bean and mung bean inhibit the antigen specific activation of helper T cells by blocking cell cycle progression.

Science.gov (United States)

Lee, Suk Jun; Bae, Joonbeom; Kim, Sunhee; Jeong, Seonah; Choi, Chang-Yong; Choi, Sang-Pil; Kim, Hyun-Sook; Jung, Woon-Won; Imm, Jee-Young; Kim, Sae Hun; Chun, Taehoon

2013-02-01

Treatment of helper T (Th) cells with saponins from soy bean and mung bean prevented their activation by inhibiting cell proliferation and cytokine secretion. However, the saponins did not affect the expression of major histocompatibility complex class II (A(b)) and co-stimulatory molecule (CD86) on professional antigen-presenting cells. Instead, the saponins directly inhibited Th cell proliferation by blocking the G(1) to S phase cell cycle transition. Moreover, blocking of the cell cycle by the saponins was achieved by decreased expression of cyclin D1 and cyclin E, and constitutive expression of p27(KIP1). Saponins also increased stability of p27(KIP1) in Th cells after antigenic stimulation.

Reduction of radiation-induced cell cycle blocks by caffeine does not necessarily lead to increased cell killing

Energy Technology Data Exchange (ETDEWEB)

Musk, S.R. (Institute of Cancer Research, Sutton, Surrey (England))

1991-03-01

The effect of caffeine upon the radiosensitivities of three human tumor lines was examined and correlated with its action upon the radiation-induced S-phase and G2-phase blocks. Caffeine was found to reduce at least partially the S-phase and G2-phase blocks in all the cell lines examined but potentiated cytotoxicity in only one of the three tumor lines. That reductions have been demonstrated to occur in the absence of increased cell killing provides supporting evidence for the hypothesis that reductions may not be causal in those cases when potentiation of radiation-induced cytotoxicity is observed in the presence of caffeine.

Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

Science.gov (United States)

Wöllert, Torsten; Langford, George M

2016-01-01

Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live

CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY IN HUMAN GLIOMA CELLS EXPOSED TO RADIATION

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Jerzy Slowinski

2011-05-01

Full Text Available Biological tests are efficient in reflecting the biological influences of several types of generally harmful exposures. The micronucleus assay is widely used in genotoxicity studies or studies on genomic damage in general. We present methodological aspects of cytokinesis-block micronucleus assay performed in human gliomas irradiated in vitro. Eight human glioblastoma cell lines obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany were gamma-irradiated (60Co over a dose range of 0-10 Gy. Cytokinesis-block micronucleus assay was performed to quantitate cytogenetic damage. The cells were fixed directly on dishes, stained with fluorochrome DAPI and evaluated under fluorescent and phase contrast microscope. The micronucleus frequency was expressed as a micronuclei (MN per binucleated cell (BNC ratio, calculated after scoring at least 100 BNC per dish. The frequency of spontaneous MN ranged from 0.17 to 0.613 (mean: 0.29 ± 0.14. After irradiation increase of MN frequency in the range of 0.312 - 2.241 (mean: 0.98 ± 0.68 was found at 10 Gy. Gliomas are extremely heterogenous in regard to cytogenetic effects of irradiation, as shown in this study by cytokinesis-block micronucleus assay. This test is easily performed on irradiated glioma cell lines and can assist in determining their radiosensitivity. However, in order to obtain reliable and reproducible results, precise criteria for MN scoring must be strictly followed. Simultaneous use of fluorescent and phase contrast equipment improves imaging of morphological details and can further optimize MN scoring.

Autofluorescence-Free Live-Cell Imaging Using Terbium Nanoparticles.

Science.gov (United States)

Cardoso Dos Santos, M; Goetz, J; Bartenlian, H; Wong, K-L; Charbonnière, L J; Hildebrandt, N

2018-04-18

Fluorescent nanoparticles (NPs) have become irreplaceable tools for advanced cellular and subcellular imaging. While very bright NPs require excitation with UV or visible light, which can create strong autofluorescence of biological components, NIR-excitable NPs without autofluorescence issues exhibit much lower brightness. Here, we show the application of a new type of surface-photosensitized terbium NPs (Tb-NPs) for autofluorescence-free intracellular imaging in live HeLa cells. The combination of exceptionally high brightness, high photostability, and long photoluminecence (PL) lifetimes for highly efficient suppression of the short-lived autofluorescence allowed for time-gated PL imaging of intracellular vesicles over 72 h without toxicity and at extremely low Tb-NP concentrations down to 12 pM. Detection of highly resolved long-lifetime (ms) PL decay curves from small (∼10 μm 2 ) areas within single cells within a few seconds emphasized the unprecedented photophysical properties of Tb-NPs for live-cell imaging that extend well beyond currently available nanometric imaging agents.

Direct and dynamic detection of HIV-1 in living cells.

Directory of Open Access Journals (Sweden)

Jonas Helma

Full Text Available In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

Imaging Proteolysis by Living Human Breast Cancer Cells

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Mansoureh Sameni

2000-01-01

Full Text Available Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549 through the use of quenchedfluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin 13-selective cysteine protease inhibitor, intracellular fluorescence was decreased ~90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence ~50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1 a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2 the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.

Creating living machines

Science.gov (United States)

Kamm, Roger D.; Bashir, Rashid

2015-01-01

Development of increasingly complex integrated cellular systems will be a major challenge for the next decade and beyond, as we apply the knowledge gained from the sub-disciplines of tissue engineering, synthetic biology, micro-fabrication and nanotechnology, systems biology, and developmental biology. In this prospective, we describe the current state-of-the-art in the context of differentiating source cells from more primitive, pluripotent cells, and organizing these cells into populations of a single cell type to produce the components or building blocks of higher order systems and finally, combining multiple cell types, possibly in combination with scaffolds possessing specific physical or chemical properties, to produce greater functionality. As these “living machines” increase in capabilities, exhibit emergent behavior and potentially reveal the ability for self-assembly, self-repair, and even self-replication, questions arise regarding the ethical implications of this work. Future prospects as well as ways of addressing these complex ethical questions will be addressed. PMID:24006130

High-frequency microrheology reveals cytoskeleton dynamics in living cells

Science.gov (United States)

Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

2017-08-01

Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

Biomimetic silica encapsultation of living cells

Science.gov (United States)

Jaroch, David Benjamin

Living cells perform complex chemical processes on size and time scales that artificial systems cannot match. Cells respond dynamically to their environment, acting as biological sensors, factories, and drug delivery devices. To facilitate the use of living systems in engineered constructs, we have developed several new approaches to create stable protective microenvironments by forming bioinspired cell-membrane-specific silica-based encapsulants. These include vapor phase deposition of silica gels, use of endogenous membrane proteins and polysaccharides as a site for silica nucleation and polycondensation in a saturated environment, and protein templated ordered silica shell formation. We demonstrate silica layer formation at the surface of pluripotent stem-like cells, bacterial biofilms, and primary murine and human pancreatic islets. Materials are characterized by AFM, SEM and EDS. Viability assays confirm cell survival, and metabolite flux measurements demonstrate normal function and no major diffusion limitations. Real time PCR mRNA analysis indicates encapsulated islets express normal levels of genetic markers for β-cells and insulin production. The silica glass encapsulant produces a secondary bone like calcium phosphate mineral layer upon exposure to media. Such bioactive materials can improve device integration with surrounding tissue upon implantation. Given the favorable insulin response, bioactivity, and long-term viability observed in silica-coated islets, we are currently testing the encapsulant's ability to prevent immune system recognition of foreign transplants for the treatment of diabetes. Such hybrid silica-cellular constructs have a wide range of industrial, environmental, and medical applications.

A hybrid bio-jetting approach for directly engineering living cells

International Nuclear Information System (INIS)

Kwok, Albert; Irvine, Scott; Arumuganathar, Sumathy; Jayasinghe, Suwan N; McEwan, Jean R

2008-01-01

This paper reports developments on a hybrid cell-engineering protocol coupling both bio-electrosprays and aerodynamically assisted bio-jets for process-handling living cells. The current work demonstrates the ability to couple these two cell-jetting protocols for handling a wide range of cells for deposition. The post-treated cells are assessed for their viability by way of flow cytometry, which illustrates a significant population of viable cells post-treatment in comparison to those controls. This work is the first example of coupling these two protocols for the process handling of living cells. The hybrid protocol demonstrates the achievement of stable cone jetting of a cellular suspension in the single-needle configuration which was previously unachieved with single-needle bio-electrosprays. Furthermore the living cells explored in these investigations expressed GFP, thus demonstrating the ability to couple gene therapy with this hybrid protocol. Hence, this approach could one day be explored for building biologically viable tissues incorporating a therapeutic payload for combating a range of cellular/tissue-based pathologies

Validation of 31 of the most commonly used immunohistochemical antibodies in cytology prepared using the Cellient(®) automated cell block system.

Science.gov (United States)

Montgomery, Eric; Gao, Chen; de Luca, Julie; Bower, Jessie; Attwood, Kristropher; Ylagan, Lourdes

2014-12-01

The Cellient(®) cell block system has become available as an alternative, partially automated method to create cell blocks in cytology. We sought to show a validation method for immunohistochemical (IHC) staining on the Cellient cell block system (CCB) in comparison with the formalin fixed paraffin embedded traditional cell block (TCB). Immunohistochemical staining was performed using 31 antibodies on 38 patient samples for a total of 326 slides. Split samples were processed using both methods by following the Cellient(®) manufacturer's recommendations for the Cellient cell block (CCB) and the Histogel method for preparing the traditional cell block (TCB). Interpretation was performed by three pathologists and two cytotechnologists. Immunohistochemical stains were scored as: 0/1+ (negative) and 2/3+ (positive). Inter-rater agreement for each antibody was evaluated for CCB and TCB, as well as the intra-rater agreement between TCB and CCB between observers. Interobserver staining concordance for the TCB was obtained with statistical significance (P Cellient system are reliable and concordant with stains performed on the same split samples processed via a formalin fixed-paraffin embedded (FFPE) block. The Cellient system is a welcome adjunct to cytology work-flow by producing cell block material of sufficient quality to allow the use of routine IHC. © 2014 Wiley Periodicals, Inc.

Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices.

Science.gov (United States)

Chan, K L Andrew; Kazarian, Sergei G

2013-07-21

The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Science.gov (United States)

Taylor, Matthew P.; Kratchmarov, Radomir; Enquist, Lynn W.

2013-01-01

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread. PMID:23978901

Rag Deletion in Peripheral T Cells Blocks TCR Revision

Science.gov (United States)

Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

2010-01-01

Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

Nanomimics of host cell membranes block invasion and expose invasive malaria parasites.

Science.gov (United States)

Najer, Adrian; Wu, Dalin; Bieri, Andrej; Brand, Françoise; Palivan, Cornelia G; Beck, Hans-Peter; Meier, Wolfgang

2014-12-23

The fight against most infectious diseases, including malaria, is often hampered by the emergence of drug resistance and lack or limited efficacies of vaccines. Therefore, new drugs, vaccines, or other strategies to control these diseases are needed. Here, we present an innovative nanotechnological strategy in which the nanostructure itself represents the active substance with no necessity to release compounds to attain therapeutic effect and which might act in a drug- and vaccine-like dual function. Invasion of Plasmodium falciparum parasites into red blood cells was selected as a biological model for the initial validation of this approach. Stable nanomimics-polymersomes presenting receptors required for parasite attachment to host cells-were designed to efficiently interrupt the life cycle of the parasite by inhibiting invasion. A simple way to build nanomimics without postformation modifications was established. First, a block copolymer of the receptor with a hydrophobic polymer was synthesized and then mixed with a polymersome-forming block copolymer. The resulting nanomimics bound parasite-derived ligands involved in the initial attachment to host cells and they efficiently blocked reinvasion of malaria parasites after their egress from host cells in vitro. They exhibited efficacies of more than 2 orders of magnitude higher than the soluble form of the receptor, which can be explained by multivalent interactions of several receptors on one nanomimic with multiple ligands on the infective parasite. In the future, our strategy might offer interesting treatment options for severe malaria or a way to modulate the immune response.

Phenotypic characterization of mononuclear inflammatory cells following equine hydroxyapatite/collagen block grafting in rats

International Nuclear Information System (INIS)

Alsuwaiyan, Asim; Wang, Bing-Yan; Cohen, Robert E

2012-01-01

To measure the inflammatory changes associated with the implantation of an equine hydroxyapatite and collagen-containing block graft (eHAC block) in a rodent model system, an eHAC block graft was implanted subcutaneously in rats. Control groups included saline, turpentine oil, and human mineralized particulate allograft (hMPA). Animals were sacrificed and tissue samples obtained after three days, as well as after 1, 2, 4 and 8 weeks. A panel of immunologic probes was used to identify circulatory monocytic cells (ED1), resident mononuclear phagocytes (ED2), mononuclear phagocytes of lymphoid origin (ED3), expression of Ia antigen (OX6), T-cells (OX19), and B-cells (OX33). Immunocytochemical localization was performed and mononuclear cells localized with each immunologic probe counted. Rat sera obtained after eight weeks were used for nitrocellulose dot-blotting to assess circulating anti-equine immunoglobulins. Statistical analysis was performed using two-way analysis of variance, in conjunction with the Bonferroni correction to account for multiple comparisons. A transient increase in monocytes at 3 days and 1 week was observed in all groups, but was significantly higher in the turpentine control (P < 0.0001). A significant increase in the numbers of mononuclear cells detected with clones ED2 and ED3 was observed in specimens from the turpentine group, in contrast to the other groups in the 3 day to 4 week interval (P < 0.0001), as well as within all time periods (P < 0.0001). A statistically significant difference in numbers of ED3-positive cells was observed in the hMPA group compared to the saline and the eHAC block groups after one week (P < 0.0001). Significantly more OX6-positive cells were observed in the turpentine group, compared to other groups (3 days to 1 week; P < 0.0001). T-lymphocytes were essentially absent except for rats given turpentine (after 1 week). No B-lymphocyte response was found and none of the rats developed systemic anti

Functional memory B cells and long-lived plasma cells are generated after a single Plasmodium chabaudi infection in mice.

Directory of Open Access Journals (Sweden)

Francis Maina Ndungu

2009-12-01

Full Text Available Antibodies have long been shown to play a critical role in naturally acquired immunity to malaria, but it has been suggested that Plasmodium-specific antibodies in humans may not be long lived. The cellular mechanisms underlying B cell and antibody responses are difficult to study in human infections; therefore, we have investigated the kinetics, duration and characteristics of the Plasmodium-specific memory B cell response in an infection of P. chabaudi in mice. Memory B cells and plasma cells specific for the C-terminal region of Merozoite Surface Protein 1 were detectable for more than eight months following primary infection. Furthermore, a classical memory response comprised predominantly of the T-cell dependent isotypes IgG2c, IgG2b and IgG1 was elicited upon rechallenge with the homologous parasite, confirming the generation of functional memory B cells. Using cyclophosphamide treatment to discriminate between long-lived and short-lived plasma cells, we demonstrated long-lived cells secreting Plasmodium-specific IgG in both bone marrow and in spleens of infected mice. The presence of these long-lived cells was independent of the presence of chronic infection, as removal of parasites with anti-malarial drugs had no impact on their numbers. Thus, in this model of malaria, both functional Plasmodium-specific memory B cells and long-lived plasma cells can be generated, suggesting that defects in generating these cell populations may not be the reason for generating short-lived antibody responses.

Merkel cells are long-lived cells whose production is stimulated by skin injury✰

Science.gov (United States)

Wright, Margaret C.; Logan, Gregory J.; Bolock, Alexa M.; Kubicki, Adam C.; Hemphill, Julie A.; Sanders, Timothy A.; Maricich, Stephen M.

2017-01-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. PMID:27998808

Merkel cells are long-lived cells whose production is stimulated by skin injury.

Science.gov (United States)

Wright, Margaret C; Logan, Gregory J; Bolock, Alexa M; Kubicki, Adam C; Hemphill, Julie A; Sanders, Timothy A; Maricich, Stephen M

2017-02-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

Interaction between the moss Physcomitrella patens and Phytophthora: a novel pathosystem for live-cell imaging of subcellular defence.

Science.gov (United States)

Overdijk, Elysa J R; DE Keijzer, Jeroen; DE Groot, Deborah; Schoina, Charikleia; Bouwmeester, Klaas; Ketelaar, Tijs; Govers, Francine

2016-08-01

Live-cell imaging of plant-pathogen interactions is often hampered by the tissue complexity and multicell layered nature of the host. Here, we established a novel pathosystem with the moss Physcomitrella patens as host for Phytophthora. The tip-growing protonema cells of this moss are ideal for visualizing interactions with the pathogen over time using high-resolution microscopy. We tested four Phytophthora species for their ability to infect P. patens and showed that P. sojae and P. palmivora were only rarely capable to infect P. patens. In contrast, P. infestans and P. capsici frequently and successfully penetrated moss protonemal cells, showed intracellular hyphal growth and formed sporangia. Next to these successful invasions, many penetration attempts failed. Here the pathogen was blocked by a barrier of cell wall material deposited in papilla-like structures, a defence response that is common in higher plants. Another common response is the upregulation of defence-related genes upon infection and also in moss we observed this upregulation in tissues infected with Phytophthora. For more advanced analyses of the novel pathosystem we developed a special set-up that allowed live-cell imaging of subcellular defence processes by high-resolution microscopy. With this set-up, we revealed that Phytophthora infection of moss induces repositioning of the nucleus, accumulation of cytoplasm and rearrangement of the actin cytoskeleton, but not of microtubules. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Semi-automated quantification of living cells with internalized nanostructures

KAUST Repository

Margineanu, Michael B.

2016-01-15

Background Nanostructures fabricated by different methods have become increasingly important for various applications in biology and medicine, such as agents for medical imaging or cancer therapy. In order to understand their interaction with living cells and their internalization kinetics, several attempts have been made in tagging them. Although methods have been developed to measure the number of nanostructures internalized by the cells, there are only few approaches aimed to measure the number of cells that internalize the nanostructures, and they are usually limited to fixed-cell studies. Flow cytometry can be used for live-cell assays on large populations of cells, however it is a single time point measurement, and does not include any information about cell morphology. To date many of the observations made on internalization events are limited to few time points and cells. Results In this study, we present a method for quantifying cells with internalized magnetic nanowires (NWs). A machine learning-based computational framework, CellCognition, is adapted and used to classify cells with internalized and no internalized NWs, labeled with the fluorogenic pH-dependent dye pHrodo™ Red, and subsequently to determine the percentage of cells with internalized NWs at different time points. In a “proof-of-concept”, we performed a study on human colon carcinoma HCT 116 cells and human epithelial cervical cancer HeLa cells interacting with iron (Fe) and nickel (Ni) NWs. Conclusions This study reports a novel method for the quantification of cells that internalize a specific type of nanostructures. This approach is suitable for high-throughput and real-time data analysis and has the potential to be used to study the interaction of different types of nanostructures in live-cell assays.

Circumventing photodamage in live-cell microscopy

Science.gov (United States)

Magidson, Valentin; Khodjakov, Alexey

2013-01-01

Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely ‘non-invasive’ as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure. PMID:23931522

Stress-driven pattern formation in living and non-living matter

DEFF Research Database (Denmark)

Christensen, Amalie

. On the smallest scale of nanometers, we study thin films of block copolymers, which have potential applications as self-organizing templates for microelectronics. By performing a thin-shell expansion of a well-known model for block copolymers, we develop an effective model for the impact of curvature on pattern......Spatial pattern formation is abundant in nature and occurs in both living and non-living matter. Familiar examples include sand ripples, river deltas, zebra fur and snail shells. In this thesis, we focus on patterns induced by mechanical stress, and develop continuum theories for three systems...

Gaussian fluctuation of the diffusion exponent of virus capsid in a living cell nucleus

Science.gov (United States)

Itto, Yuichi

2018-05-01

In their work [4], Bosse et al. experimentally showed that virus capsid exhibits not only normal diffusion but also anomalous diffusion in nucleus of a living cell. There, it was found that the distribution of fluctuations of the diffusion exponent characterizing them takes the Gaussian form, which is, quite remarkably, the same form for two different types of the virus. This suggests high robustness of such fluctuations. Here, the statistical property of local fluctuations of the diffusion exponent of the virus capsid in the nucleus is studied. A maximum-entropy-principle approach (originally proposed for a different virus in a different cell) is applied for obtaining the fluctuation distribution of the exponent. Largeness of the number of blocks identified with local areas of interchromatin corrals is also examined based on the experimental data. It is shown that the Gaussian distribution of the local fluctuations can be derived, in accordance with the above form. In addition, it is quantified how the fluctuation distribution on a long time scale is different from the Gaussian distribution.

Rolling block mazes are PSPACE-complete

NARCIS (Netherlands)

Buchin, K.; Buchin, M.

2012-01-01

In a rolling block maze, one or more blocks lie on a rectangular board with square cells. In most mazes, the blocks have size k × m × n where k, m, n are integers that determine the size of the block in terms of units of the size of the board cells. The task of a rolling block maze is to roll a

Acid base activity of live bacteria: Implications for quantifying cell wall charge

Science.gov (United States)

Claessens, Jacqueline; van Lith, Yvonne; Laverman, Anniet M.; Van Cappellen, Philippe

2006-01-01

To distinguish the buffering capacity associated with functional groups in the cell wall from that resulting from metabolic processes, base or acid consumption by live and dead cells of the Gram-negative bacterium Shewanella putrefaciens was measured in a pH stat system. Live cells exhibited fast consumption of acid (pH 4) or base (pH 7, 8, 9, and 10) during the first few minutes of the experiments. At pH 5.5, no acid or base was required to maintain the initial pH constant. The initial amounts of acid or base consumed by the live cells at pH 4, 8, and 10 were of comparable magnitudes as those neutralized at the same pHs by intact cells killed by exposure to gamma radiation or ethanol. Cells disrupted in a French press required higher amounts of acid or base, due to additional buffering by intracellular constituents. At pH 4, acid neutralization by suspensions of live cells stopped after 50 min, because of loss of viability. In contrast, under neutral and alkaline conditions, base consumption continued for the entire duration of the experiments (5 h). This long-term base neutralization was, at least partly, due to active respiration by the cells, as indicated by the build-up of succinate in solution. Qualitatively, the acid-base activity of live cells of the Gram-positive bacterium Bacillus subtilis resembled that of S. putrefaciens. The pH-dependent charging of ionizable functional groups in the cell walls of the live bacteria was estimated from the initial amounts of acid or base consumed in the pH stat experiments. From pH 4 to 10, the cell wall charge increased from near-zero values to about -4 × 10 -16 mol cell -1 and -6.5 × 10 -16 mol cell -1 for S. putrefaciens and B. subtilis, respectively. The similar cell wall charging of the two bacterial strains is consistent with the inferred low contribution of lipopolysaccharides to the buffering capacity of the Gram-negative cell wall (of the order of 10%).

Axial tomography in 3D live cell microscopy

Science.gov (United States)

Richter, Verena; Bruns, Sarah; Bruns, Thomas; Piper, Mathis; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

2017-07-01

A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.

Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

Directory of Open Access Journals (Sweden)

Neil O Carragher

2011-04-01

Full Text Available Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates.

The effects of atomic force microscopy upon nominated living cells

Energy Technology Data Exchange (ETDEWEB)

O' Hagan, Barry Michael Gerard [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom)]. E-mail: bmg.ohagan@ulstser.ac.uk; Doyle, Peter [Unilever Research, Port Sunlight, The Wirral, Merseyside (United Kingdom); Allen, James M. [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom); Sutton, Kerry [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom); McKerr, George [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom)

2004-12-15

This work describes a system for precise re-location of cells within a monolayer after atomic force imaging. As we know little about probe interaction with soft biological surfaces any corroborative evidence is of great importance. For example, it is of paramount importance in living cell force microscopy that interrogated cells can be re-located and imaged by other corroborative technologies. Methodologies expressed here have shown that non-invasive force parameters can be established for specific cell types. Additionally, we show that the same sample can be transferred reliably to an SEM. Results here indicate that further work with live cells should initially establish appropriate prevailing force parameters and that cell damage should be checked for before and after an imaging experiment.

The effects of atomic force microscopy upon nominated living cells

International Nuclear Information System (INIS)

O'Hagan, Barry Michael Gerard; Doyle, Peter; Allen, James M.; Sutton, Kerry; McKerr, George

2004-01-01

This work describes a system for precise re-location of cells within a monolayer after atomic force imaging. As we know little about probe interaction with soft biological surfaces any corroborative evidence is of great importance. For example, it is of paramount importance in living cell force microscopy that interrogated cells can be re-located and imaged by other corroborative technologies. Methodologies expressed here have shown that non-invasive force parameters can be established for specific cell types. Additionally, we show that the same sample can be transferred reliably to an SEM. Results here indicate that further work with live cells should initially establish appropriate prevailing force parameters and that cell damage should be checked for before and after an imaging experiment

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

Science.gov (United States)

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

HaloTag protein-mediated specific labeling of living cells with quantum dots

International Nuclear Information System (INIS)

So, Min-kyung; Yao Hequan; Rao Jianghong

2008-01-01

Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging

[Development of a Fluorescence Probe for Live Cell Imaging].

Science.gov (United States)

Shibata, Aya

2017-01-01

 Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19 F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19 F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

Proliferative and Invasive Effects of Progesterone-Induced Blocking Factor in Human Glioblastoma Cells

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Araceli Gutiérrez-Rodríguez

2017-01-01

Full Text Available Progesterone-induced blocking factor (PIBF is a progesterone (P4 regulated protein expressed in different types of high proliferative cells including astrocytomas, the most frequent and aggressive brain tumors. It has been shown that PIBF increases the number of human astrocytoma cells. In this work, we evaluated PIBF regulation by P4 and the effects of PIBF on proliferation, migration, and invasion of U87 and U251 cells, both derived from human glioblastomas. PIBF mRNA expression was upregulated by P4 (10 nM from 12 to 24 h. Glioblastoma cells expressed two PIBF isoforms, 90 and 57 kDa. The content of the shorter isoform was increased by P4 at 24 h, while progesterone receptor antagonist RU486 (10 μM blocked this effect. PIBF (100 ng/mL increased the number of U87 cells on days 4 and 5 of treatment and induced cell proliferation on day 4. Wound-healing assays showed that PIBF increased the migration of U87 (12–48 h and U251 (24 and 48 h cells. Transwell invasion assays showed that PIBF augmented the number of invasive cells in both cell lines at 24 h. These data suggest that PIBF promotes proliferation, migration, and invasion of human glioblastoma cells.

Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

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Hempstead, Andrew D; Isberg, Ralph R

2015-12-08

Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

Science.gov (United States)

Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

2016-11-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

Cyborg cells: functionalisation of living cells with polymers and nanomaterials.

Science.gov (United States)

Fakhrullin, Rawil F; Zamaleeva, Alsu I; Minullina, Renata T; Konnova, Svetlana A; Paunov, Vesselin N

2012-06-07

Living cells interfaced with a range of polyelectrolyte coatings, magnetic and noble metal nanoparticles, hard mineral shells and other complex nanomaterials can perform functions often completely different from their original specialisation. Such "cyborg cells" are already finding a range of novel applications in areas like whole cell biosensors, bioelectronics, toxicity microscreening, tissue engineering, cell implant protection and bioanalytical chemistry. In this tutorial review, we describe the development of novel methods for functionalisation of cells with polymers and nanoparticles and comment on future advances in this technology in the light of other literature approaches. We review recent studies on the cell viability and function upon direct deposition of nanoparticles, coating with polyelectrolytes, polymer assisted assembly of nanomaterials and hard shells on the cell surface. The cell toxicity issues are considered for many practical applications in terms of possible adverse effects of the deposited polymers, polyelectrolytes and nanoparticles on the cell surface.

Cell tracking using iron oxide fails to distinguish dead from living transplanted cells in the infarcted heart.

Science.gov (United States)

Winter, E M; Hogers, B; van der Graaf, L M; Gittenberger-de Groot, A C; Poelmann, R E; van der Weerd, L

2010-03-01

Recently, debate has arisen about the usefulness of cell tracking using iron oxide-labeled cells. Two important issues in determining the usefulness of cell tracking with MRI are generally overlooked; first, the effect of graft rejection in immunocompetent models, and second, the necessity for careful histological confirmation of the fate of the labeled cells in the presence of iron oxide. Therefore, both iron oxide-labeled living as well as dead epicardium-derived cells (EPDCs) were investigated in ischemic myocardium of immunodeficient non-obese diabetic (NOD)/acid: non-obese diabetic severe combined immunodeficient (NOD/scid) mice with 9.4T MRI until 6 weeks after surgery, at which time immunohistochemical analysis was performed. In both groups, voids on MRI scans were observed that did not change in number, size, or localization over time. Based on MRI, no distinction could be made between living and dead injected cells. Prussian blue staining confirmed that the hypointense spots on MRI corresponded to iron-loaded cells. However, in the dead-EPDC recipients, all iron-positive cells appeared to be macrophages, while the living-EPDC recipients also contained engrafted iron-loaded EPDCs. Iron labeling is inadequate for determining the fate of transplanted cells in the immunodeficient host, since dead cells produce an MRI signal indistinguishable from incorporated living cells. (c) 2010 Wiley-Liss, Inc.

Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

Directory of Open Access Journals (Sweden)

Akira C Saito

Full Text Available Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC field to induce a linear cell-GUV alignment, and then a direct current (DC pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.

Temperature-dependent imaging of living cells by AFM

International Nuclear Information System (INIS)

Espenel, Cedric; Giocondi, Marie-Cecile; Seantier, Bastien; Dosset, Patrice; Milhiet, Pierre-Emmanuel; Le Grimellec, Christian

2008-01-01

Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 deg. C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature

Teachable, high-content analytics for live-cell, phase contrast movies.

Science.gov (United States)

Alworth, Samuel V; Watanabe, Hirotada; Lee, James S J

2010-09-01

CL-Quant is a new solution platform for broad, high-content, live-cell image analysis. Powered by novel machine learning technologies and teach-by-example interfaces, CL-Quant provides a platform for the rapid development and application of scalable, high-performance, and fully automated analytics for a broad range of live-cell microscopy imaging applications, including label-free phase contrast imaging. The authors used CL-Quant to teach off-the-shelf universal analytics, called standard recipes, for cell proliferation, wound healing, cell counting, and cell motility assays using phase contrast movies collected on the BioStation CT and BioStation IM platforms. Similar to application modules, standard recipes are intended to work robustly across a wide range of imaging conditions without requiring customization by the end user. The authors validated the performance of the standard recipes by comparing their performance with truth created manually, or by custom analytics optimized for each individual movie (and therefore yielding the best possible result for the image), and validated by independent review. The validation data show that the standard recipes' performance is comparable with the validated truth with low variation. The data validate that the CL-Quant standard recipes can provide robust results without customization for live-cell assays in broad cell types and laboratory settings.

Fourier analysis of cell-wise Block-Jacobi splitting in two-dimensional geometry

International Nuclear Information System (INIS)

Rosa, M.; Warsa, J. S.; Kelley, T. M.

2009-01-01

A Fourier analysis is conducted in two-dimensional (2D) geometry for the discrete ordinates (S N ) approximation of the neutron transport problem solved with Richardson iteration (Source Iteration) using the cell-wise Block-Jacobi (BJ) algorithm. The results of the Fourier analysis show that convergence of cell-wise BJ can degrade, leading to a spectral radius equal to 1, in problems containing optically thin cells. For problems containing cells that are optically thick, instead, the spectral radius tends to 0. Hence, in the optically thick-cell regime, cell-wise BJ is rapidly convergent even for problems that are scattering dominated, with a scattering ratio c close to 1. (authors)

Quantification of nanowire uptake by live cells

KAUST Repository

Margineanu, Michael B.

2015-01-01

attempts have been made at tagging and investigating their interaction with living cells. In this study, magnetic iron nanowires with an iron oxide layer are coated with (3-Aminopropyl)triethoxysilane (APTES), and subsequently labeled with a fluorogenic p

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

Science.gov (United States)

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Biological interaction of living cells with COSAN-based synthetic vesicles.

Science.gov (United States)

Tarrés, Màrius; Canetta, Elisabetta; Paul, Eleanor; Forbes, Jordan; Azzouni, Karima; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J

2015-01-15

Cobaltabisdicarbollide (COSAN) [3,3'-Co(1,2-C2B9H11)2](-), is a complex boron-based anion that has the unusual property of self-assembly into membranes and vesicles. These membranes have similar dimensions to biological membranes found in cells, and previously COSAN has been shown to pass through synthetic lipid membranes and those of living cells without causing breakdown of membrane barrier properties. Here, we investigate the interaction of this inorganic membrane system with living cells. We show that COSAN has no immediate effect on cell viability, and cells fully recover when COSAN is removed following exposure for hours to days. COSAN elicits a range of cell biological effects, including altered cell morphology, inhibition of cell growth and, in some cases, apoptosis. These observations reveal a new biology at the interface between inorganic, synthetic COSAN membranes and naturally occurring biological membranes.

THE NISSL SUBSTANCE OF LIVING AND FIXED SPINAL GANGLION CELLS

Science.gov (United States)

Deitch, Arline D.; Moses, Montrose J.

1957-01-01

Living chick spinal ganglion neurons grown for 19 to 25 days in vitro were photographed with a color-translating ultraviolet microscope (UV-91) at 265, 287, and 310 mµ. This instrument was unique in permitting rapid accumulation of ultraviolet information with minimal damage to the cell. In the photographs taken at 265 mµ of the living neurons, discrete ultraviolet-absorbing cytoplasmic masses were observed which were found to be virtually unchanged in appearance after formalin fixation. These were identical with the Nissl bodies of the same cells seen after staining with basic dyes. The correlation of ultraviolet absorption, ribonuclease extraction, and staining experiments with acid and basic dyes confirmed the ribonucleoprotein nature of these Nissl bodies in the living and fixed cells. No change in distribution or concentration of ultraviolet-absorbing substance was observed in the first 12 ultraviolet photographs of a neuron, and it is concluded that the cells had not been subjected to significant ultraviolet damage during the period of photography. On the basis of these observations, as well as previous findings with phase contrast microscopy, it is concluded that Nissl bodies preexist in the living neuron as discrete aggregates containing high concentrations of nucleoprotein. PMID:13438929

Dismantling of a hot cell-block and the treatment of the produced concrete bars

International Nuclear Information System (INIS)

Rompf, U.; Brielmayer, M.; Graf, A.; Stutz, U.; Ambos, F.

2003-01-01

A building with hot cells had been operated in Karlstein/Main from 1968 to 1989 in order to perform check-ups at radiated fuel rods and nuclear components. The operation of the system was stopped after an operation period of approximately 20 years. The core part of the building to be disassembled is a U-shaped hot cell-block with nine individual cells, partly consisting of heavy reinforced concrete, located in the ground floor (fig. 1 and fig. 2). The major part of the cells was covered with 10 mm steel plate and provided with approx. 1,400 openings of all different kinds. The wall thickness of the cells was between 0.90 m and 1.10 m. Under these conditions a successful decontamination at the ''existing building structure'' was not possible. Therefore, the non-supporting structures of the hot cell-block were removed in individual blocks by means of sawing and the remaining walls and floors were peeled by using the diamond rope sawing technique. The dismantling took 17 months. A re-treatment of the produced concrete blocks (235 blocks, approx. 970 Mg) to reduce the radioactive waste to a minimum was performed at the Research Centre Karlsruhe, Central Decontamination Department (HDB). The Target of the concrete bar treatment at HDB is to reduce the volume of radioactive waste to a minimum and to add the major part of the concrete bars to harmless utilisation. To achieve the same, initially the more contaminated parts of the bars without openings, such as tubes, cable or ventilating shafts, are removed by means of wire cutting and packed into a KONRAD-Container as radioactive waste. The remaining bar is decontaminated by means of sandblasting and afterwards, following successful release measurement, released from the scope of the regulations under the Atomic Energy. Bars with openings are crushed into small pieces by means of the remote-controlled chisel excavator, in order to separate the individual kinds of material. The rubble is packed into drums and measured by

Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223

Directory of Open Access Journals (Sweden)

Weeks Sara K

2009-01-01

Full Text Available Abstract Background The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The inc gene CT223 is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested C. trachomatis serovars. Results A plasmid transfection approach was used to examine the function of the product of CT223 and other Inc proteins within uninfected mammalian cells. Fluorescence microscopy was used to demonstrate that CT223, and, to a lesser extent, adjacent inc genes, are capable of blocking host cell cytokinesis and facilitating centromere supranumeracy defects seen by others in chlamydiae-infected cells. Both phenotypes were associated with transfection of plasmids encoding the carboxy-terminal tail of CT223p, a region of the protein that is likely exposed to the cytosol in infected cells. Conclusion These studies suggest that certain Inc proteins block cytokinesis in C. trachomatis-infected cells. These results are consistent with the work of others showing chlamydial inhibition of host cell cytokinesis.

Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

Science.gov (United States)

König, Alexander; Glebe, Dieter

2017-01-01

To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

Ultrafast nanolaser device for detecting cancer in a single live cell.

Energy Technology Data Exchange (ETDEWEB)

Gourley, Paul Lee; McDonald, Anthony Eugene

2007-11-01

Emerging BioMicroNanotechnologies have the potential to provide accurate, realtime, high throughput screening of live tumor cells without invasive chemical reagents when coupled with ultrafast laser methods. These optically based methods are critical to advancing early detection, diagnosis, and treatment of disease. The first year goals of this project are to develop a laser-based imaging system integrated with an in- vitro, live-cell, micro-culture to study mammalian cells under controlled conditions. In the second year, the system will be used to elucidate the morphology and distribution of mitochondria in the normal cell respiration state and in the disease state for normal and disease states of the cell. In this work we designed and built an in-vitro, live-cell culture microsystem to study mammalian cells under controlled conditions of pH, temp, CO2, Ox, humidity, on engineered material surfaces. We demonstrated viability of cell culture in the microsystem by showing that cells retain healthy growth rates, exhibit normal morphology, and grow to confluence without blebbing or other adverse influences of the material surfaces. We also demonstrated the feasibility of integrating the culture microsystem with laser-imaging and performed nanolaser flow spectrocytometry to carry out analysis of the cells isolated mitochondria.

Impact of mobility on call block, call drops and optimal cell size in small cell networks

OpenAIRE

Ramanath , Sreenath; Voleti , Veeraruna Kavitha; Altman , Eitan

2011-01-01

We consider small cell networks and study the impact of user mobility. Assuming Poisson call arrivals at random positions with random velocities, we discuss the characterization of handovers at the boundaries. We derive explicit expressions for call block and call drop probabilities using tools from spatial queuing theory. We also derive expressions for the average virtual server held up time. These expressions are used to derive optimal cell sizes for various profile of velocities in small c...

Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging

Directory of Open Access Journals (Sweden)

Feng Gao

2016-08-01

Full Text Available Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag, by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.

Rewiring cells: synthetic biology as a tool to interrogate the organizational principles of living systems.

Science.gov (United States)

Bashor, Caleb J; Horwitz, Andrew A; Peisajovich, Sergio G; Lim, Wendell A

2010-01-01

The living cell is an incredibly complex entity, and the goal of predictively and quantitatively understanding its function is one of the next great challenges in biology. Much of what we know about the cell concerns its constituent parts, but to a great extent we have yet to decode how these parts are organized to yield complex physiological function. Classically, we have learned about the organization of cellular networks by disrupting them through genetic or chemical means. The emerging discipline of synthetic biology offers an additional, powerful approach to study systems. By rearranging the parts that comprise existing networks, we can gain valuable insight into the hierarchical logic of the networks and identify the modular building blocks that evolution uses to generate innovative function. In addition, by building minimal toy networks, one can systematically explore the relationship between network structure and function. Here, we outline recent work that uses synthetic biology approaches to investigate the organization and function of cellular networks, and describe a vision for a synthetic biology toolkit that could be used to interrogate the design principles of diverse systems.

Understanding of Protein Synthesis in a Living Cell

Science.gov (United States)

Mustapha, Y.; Muhammad, S.

2006-01-01

The assembly of proteins takes place in the cytoplasm of a cell. There are three main steps. In initiation, far left, all the necessary parts of the process are brought together by a small molecule called a ribosome. During elongation, amino acids, the building blocks of proteins, are joined to one another in a long chain. The sequence in which…

Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

Science.gov (United States)

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

2010-01-01

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

Science.gov (United States)

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

2009-11-26

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

Live-cell super-resolution imaging of intrinsically fast moving flagellates

International Nuclear Information System (INIS)

Glogger, M; Subota, I; Spindler, M-C; Engstler, M; Fenz, S F; Stichler, S; Bertlein, S; Teßmar, J; Groll, J

2017-01-01

Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μ s. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. (paper)

Uranium and thorium uptake by live and dead cells of Pseudomonas Sp

International Nuclear Information System (INIS)

Siva Prasath, C.S.; Manikandan, N.; Prakash, S.

2010-01-01

This study presents uptake of uranium (U) and thorium (Th) by live and dead cells of Pseudomonas Sp. Increasing concentration of U and Tb showed decrease in absorption by Pseudomonas Sp. Dead cells of Pseudomonas Sp. exhibited same or more uptake of U and Th than living cells. Increasing temperature promotes uptake of U and Th by Pseudomonas Sp. (author)

UV light blocks EGFR signalling in human cancer cell lines

DEFF Research Database (Denmark)

Olsen, BB; Neves-Petersen, M T; Klitgaard, S

2007-01-01

UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...... antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin...... disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV light treatment....

Accurate live and dead bacterial cell enumeration using flow cytometry (Conference Presentation)

Science.gov (United States)

Ou, Fang; McGoverin, Cushla; Swift, Simon; Vanholsbeeck, Frédérique

2017-03-01

Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with particular characteristics of interest. However most FCM cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. The easiest, most reliable and inexpensive way of obtaining absolute counts with FCM is by using reference beads. We investigated a method of using FCM with reference beads to measure live and dead bacterial concentration over the range of 106 to 108 cells/mL and ratio varying from 0 to 100%. We believe we are the first to use this method for such a large cell concentration range while also establishing the effect of varying the live/dead bacteria ratios. Escherichia coli solutions with differing ratios of live:dead cells were stained with fluorescent dyes SYTO 9 and propidium iodide (PI), which label live and dead cells, respectively. Samples were measured using a LSR II Flow Cytometer (BD Biosciences); using 488 nm excitation with 20 mW power. Both SYTO 9 and PI fluorescence were collected and threshold was set to side scatter. Traditional culture-based plate count was done in parallel to the FCM analysis. The concentration of live bacteria from FCM was compared to that obtained by plate counts. Preliminary results show that the concentration of live bacteria obtained by FCM and plate counts correlate well with each other and indicates this may be extended to a wider concentration range or for studying other cell characteristics.

Contribution of ultrasound-guided fine-needle aspiration cell blocks of metastatic supraclavicular lymph nodes to the diagnosis of lung cancer

Directory of Open Access Journals (Sweden)

Hai-Ying Tian

2015-01-01

Conclusion: Cell-block samples from US-guided FNA is a promising, relatively noninvasive technique to provide additional information in lung cancer diagnosis. Analysis of cell blocks allows for genetic analysis of the patients with supraclavicular lymph nodes metastasis.

FORMING SELF-ASSEMBLED CELL ARRAYS AND MEASURING THE OXYGEN CONSUMPTION RATE OF A SINGLE LIVE CELL.

Science.gov (United States)

Etzkorn, James R; McQuaide, Sarah C; Anderson, Judy B; Meldrum, Deirdre R; Parviz, Babak A

2009-06-01

We report a method for forming arrays of live single cells on a chip using polymer micro-traps made of SU8. We have studied the toxicity of the microfabricated structures and the associated environment for two cell lines. We also report a method for measuring the oxygen consumption rate of a single cell using optical interrogation of molecular oxygen sensors placed in micromachined micro-wells by temporarily sealing the cells in the micro-traps. The new techniques presented here add to the collection of tools available for performing "single-cell" biology. A single-cell self-assembly yield of 61% was achieved with oxygen draw down rates of 0.83, 0.82, and 0.71 fmol/minute on three isolated live A549 cells.

Modulation of radiation-induced apoptosis and G2/M block in murine T-lymphoma cells

International Nuclear Information System (INIS)

Palayoor, S.T.; Macklis, R.M.; Bump, E.A.; Coleman, C.N.

1995-01-01

Radiation-induced apoptosis in lymphocyte-derived cell lines is characterized by endonucleolytic cleavage of cellular DNA within hours after radiation exposure. We have studied this phenomenon qualitatively (DNA gel electrophoresis) and quantitatively (diphenylamine reagent assay) in murine EL4 T-lymphoma cells exposed to 137 Cs γ irradiation. Fragmentation was discernible within 18-24 h after exposure. It increased with time and dose and reached a plateau after 8 Gy of γ radiation. We studied the effect of several pharmacological agents on the radiation-induced G 2 /M block and DNA fragmentation. The agents which reduced the radiation-induced G 2 /M-phase arrest (caffeine, theobromine, theophylline and 2-aminopurine) enhanced the degree of DNA fragmentation at 24 h. In contrast, the agents which sustained the radiation-induced G 2 /M-phase arrest (TPA, DBcAMP, IBMX and 3-aminobenzamide) inhibited the DNA fragmentation at 24 h. These studies on EL4 lymphoma cells are consistent with the hypothesis that cells with radiation-induced genetic damage are eliminated by apoptosis subsequent to a G 2 /M block. Furthermore, it may be possible to modulate the process of radiation-induced apoptosis in lymphoma cells with pharmacological agents that modify the radiation-induced G 2 /M block, and to use this effect in the treatment of patients with malignant disease. 59 refs., 7 figs

Background fluorescence estimation and vesicle segmentation in live cell imaging with conditional random fields.

Science.gov (United States)

Pécot, Thierry; Bouthemy, Patrick; Boulanger, Jérôme; Chessel, Anatole; Bardin, Sabine; Salamero, Jean; Kervrann, Charles

2015-02-01

Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

Effect of transversus abdominis plane block in combination with general anesthesia on perioperative opioid consumption, hemodynamics, and recovery in living liver donors: The prospective, double-blinded, randomized study.

Science.gov (United States)

Erdogan, Mehmet A; Ozgul, Ulku; Uçar, Muharrem; Yalin, Mehmet R; Colak, Yusuf Z; Çolak, Cemil; Toprak, Huseyin I

2017-04-01

Transversus abdominis plane (TAP) block provides effective postoperative analgesia after abdominal surgeries. It can be also a useful strategy to reduce perioperative opioid consumption, support intraoperative hemodynamic stability, and promote early recovery from anesthesia. The aim of this prospective randomized double-blind study was to assess the effect of subcostal TAP blocks on perioperative opioid consumption, hemodynamic, and recovery time in living liver donors. The prospective, double-blinded, randomized controlled study was conducted with 49 living liver donors, aged 18-65 years, who were scheduled to undergo right hepatectomy. Patients who received subcostal TAP block in combination with general anesthesia were allocated into Group 1, and patients who received general anesthesia alone were allocated into Group 2. The TAP blocks were performed bilaterally by obtaining an image with real-time ultrasound guidance using 0.5% bupivacaine diluted with saline to reach a total volume of 40 mL. The primary outcome measure in our study was perioperative remifentanil consumption. Secondary outcomes were mean blood pressure (MBP), heart rate (HR), mean desflurane requirement, anesthesia recovery time, frequency of emergency vasopressor use, total morphine use, and length of hospital stay. Total remifentanil consumption and the anesthesia recovery time were significantly lower in Group 1 compared with Group 2. Postoperative total morphine use and length of hospital stay were also reduced. Changes in the MAP and HR were similar in the both groups. There were no significant differences in HR and MBP between groups at any time. Combining subcostal TAP blocks with general anesthesia significantly reduced perioperative and postoperative opioid consumption, provided shorter anesthesia recovery time, and length of hospital stay in living liver donors. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Label-free and live cell imaging by interferometric scattering microscopy.

Science.gov (United States)

Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

2018-03-14

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

Collective Dynamics of Intracellular Water in Living Cells

International Nuclear Information System (INIS)

Orecchini, A; Sebastiani, F; Paciaroni, A; Petrillo, C; Sacchetti, F; Jasnin, M; Francesco, A De; Zaccai, G; Moulin, M; Haertlein, M

2012-01-01

Water dynamics plays a fundamental role for the fulfillment of biological functions in living organisms. Decades of hydrated protein powder studies have revealed the peculiar dynamical properties of hydration water with respect to pure water, due to close coupling interactions with the macromolecule. In such a framework, we have studied coherent collective dynamics in protein and DNA hydration water. State-of-the-art neutron instrumentation has allowed us to observe the propagation of coherent density fluctuations within the hydration shell of the biomolecules. The corresponding dispersion curves resulted to be only slightly affected by the coupling with the macromolecules. Nevertheless, the effects of the interaction appeared as a marked increase of the mode damping factors, which suggested a destructuring of the water hydrogen-bond network. Such results were interpreted as the signature of a 'glassy' dynamical character of macromolecule hydration water, in agreement with indications from measurements of the density of vibrational states. Extending the investigations to living organisms at physiological conditions, we present here an in-vivo study of collective dynamics of intracellular water in Escherichia coli cells. The cells and water were fully deuterated to minimise the incoherent neutron scattering background. The water dynamics observed in the living cells is discussed in terms of the dynamics of pure bulk water and that of hydration water measured in powder samples.

Picosecond orientational dynamics of water in living cells.

Science.gov (United States)

Tros, Martijn; Zheng, Linli; Hunger, Johannes; Bonn, Mischa; Bonn, Daniel; Smits, Gertien J; Woutersen, Sander

2017-10-12

Cells are extremely crowded, and a central question in biology is how this affects the intracellular water. Here, we use ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy to observe the random orientational motion of water molecules inside living cells of three prototypical organisms: Escherichia coli, Saccharomyces cerevisiae (yeast), and spores of Bacillus subtilis. In all three organisms, most of the intracellular water exhibits the same random orientational motion as neat water (characteristic time constants ~9 and ~2 ps for the first-order and second-order orientational correlation functions), whereas a smaller fraction exhibits slower orientational dynamics. The fraction of slow intracellular water varies between organisms, ranging from ~20% in E. coli to ~45% in B. subtilis spores. Comparison with the water dynamics observed in solutions mimicking the chemical composition of (parts of) the cytosol shows that the slow water is bound mostly to proteins, and to a lesser extent to other biomolecules and ions.The cytoplasm's crowdedness leads one to expect that cell water is different from bulk water. By measuring the rotational motion of water molecules in living cells, Tros et al. find that apart from a small fraction of water solvating biomolecules, cell water has the same dynamics as bulk water.

Electrochemical Characterization of TiO 2 Blocking Layers for Dye-Sensitized Solar Cells

KAUST Repository

Kavan, Ladislav

2014-07-31

Thin compact layers of TiO2 are grown by thermal oxidation of Ti, by spray pyrolysis, by electrochemical deposition, and by atomic layer deposition. These layers are used in dye-sensitized solar cells to prevent recombination of electrons from the substrate (FTO or Ti) with the hole-conducting medium at this interface. The quality of blocking is evaluated electrochemically by methylviologen, ferro/ferricyanide, and spiro-OMeTAD as the model redox probes. Two types of pinholes in the blocking layers are classified, and their effective area is quantified. Frequency-independent Mott-Schottky plots are fitted from electrochemical impedance spectroscopy. Certain films of the thicknesses of several nanometers allow distinguishing the depletion layer formation both in the TiO2 film and in the FTO substrate underneath the titania film. The excellent blocking function of thermally oxidized Ti, electrodeposited film (60 nm), and atomic-layer-deposited films (>6 nm) is documented by the relative pinhole area of less than 1%. However, the blocking behavior of electrodeposited and atomic-layer-deposited films is strongly reduced upon calcination at 500 °C. The blocking function of spray-pyrolyzed films is less good but also less sensitive to calcination. The thermally oxidized Ti is well blocking and insensitive to calcination. © 2014 American Chemical Society.

Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

Science.gov (United States)

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

Live-cell thermometry with nitrogen vacancy centers in nanodiamonds

Science.gov (United States)

Jayakumar, Harishankar; Fedder, Helmut; Chen, Andrew; Yang, Liudi; Li, Chenghai; Wrachtrup, Joerg; Wang, Sihong; Meriles, Carlos

The ability to measure temperature is typically affected by a tradeoff between sensitivity and spatial resolution. Good thermometers tend to be bulky systems and hence are ill-suited for thermal sensing with high spatial localization. Conversely, the signal resulting from nanoscale temperature probes is often impacted by noise to a level where the measurement precision becomes poor. Adding to the microscopist toolbox, the nitrogen vacancy (NV) center in diamond has recently emerged as a promising platform for high-sensitivity nanoscale thermometry. Of particular interest are applications in living cells because diamond nanocrystals are biocompatible and can be chemically functionalized to target specific organelles. Here we report progress on the ability to probe and compare temperature within and between living cells using nanodiamond-hosted NV thermometry. We focus our study on cancerous cells, where atypical metabolic pathways arguably lead to changes in the way a cell generates heat, and thus on its temperature profile.

Anti-S100A4 antibody suppresses metastasis formation by blocking stroma cell invasion

DEFF Research Database (Denmark)

Klingelhöfer, Jörg; Grum-Schwensen, Birgitte; Beck, Mette K

2012-01-01

microenvironment, making it an attractive target for anti-cancer therapy. In this study, we produced a function-blocking anti-S100A4 monoclonal antibody with metastasis-suppressing activity. Antibody treatment significantly reduced metastatic burden in the lungs of experimental animals by blocking the recruitment...... of T cells to the site of the primary tumor. In vitro studies demonstrated that this antibody efficiently reduced the invasion of T cells in a fibroblast monolayer. Moreover, it was capable of suppressing the invasive growth of human and mouse fibroblasts. We presume therefore that the antibody exerts...... its activity by suppressing stroma cell recruitment to the site of the growing tumor. Our epitope mapping studies suggested that the antibody recognition site overlaps with the target binding interface of human S100A4. We conclude here that this antibody could serve as a solid basis for development...

Comparison of the effectiveness and safety between lymphocytes scavenger and IL-2 receptor blocking agent induction in living kidney transplantation

Directory of Open Access Journals (Sweden)

Ning-bo QIN

2013-03-01

Full Text Available Objective 　To compare the safety of two antibody inductors, namely lymphocytes scavenger and IL-2 receptor blocking agent, in living kidney transplantation. Methods 　The data of 191 patients, who received living kidney transplant in our hospital from Feb. 2007 to Jul. 2012, were retrospectively analyzed, and grouped according to the inductors they received as: a lymphocytes scavenger group (n=56, with rabbit antithymocyte immunoglobulin (rATG, 4 cases and porcine antihuman T-lymphocyte immunoglobulin (pATG, 52 cases served as the inductor; b IL -2 receptor blocking agent group (n=54, with basiliximab (40 cases and daclizumab (14 cases served as the inductor; and c control group (n=81. The incidence of rejection and infection, and the survival rate of patient/allograft within one year were then compared among the three groups. Results 　Within one year after the transplantation, the incidence of acute rejection in lymphocytes scavenger group, IL-2 receptor blocking agent group and control group was 12.5%, 11.1% and 28.4%, respectively. There was a significant difference between the two inductor groups and control group (P=0.003, but no significant difference was found between the two inductor groups (P>0.05. The incidence of delayed graft function (DGF in the three groups was 8.9%, 7.4% and 13.6%, respectively, with no statistical significance (P>0.05. Also there was no significant difference among the three groups in the incidence of infection and the survival rate of patient/allograft within one year after transplantation (P>0.05. Conclusion 　Both inductors may significantly reduce the incidence of acute rejection within one year without increasing the incidence of infection and other adverse events, nor affect the postoperative patient/graft survival, so they are both safe and effective.

Retinal ganglion cells: mechanisms underlying depolarization block and differential responses to high frequency electrical stimulation of ON and OFF cells

Science.gov (United States)

Kameneva, T.; Maturana, M. I.; Hadjinicolaou, A. E.; Cloherty, S. L.; Ibbotson, M. R.; Grayden, D. B.; Burkitt, A. N.; Meffin, H.

2016-02-01

Objective. ON and OFF retinal ganglion cells (RGCs) are known to have non-monotonic responses to increasing amplitudes of high frequency (2 kHz) biphasic electrical stimulation. That is, an increase in stimulation amplitude causes an increase in the cell’s spike rate up to a peak value above which further increases in stimulation amplitude cause the cell to decrease its activity. The peak response for ON and OFF cells occurs at different stimulation amplitudes, which allows differential stimulation of these functional cell types. In this study, we investigate the mechanisms underlying the non-monotonic responses of ON and OFF brisk-transient RGCs and the mechanisms underlying their differential responses. Approach. Using in vitro patch-clamp recordings from rat RGCs, together with simulations of single and multiple compartment Hodgkin-Huxley models, we show that the non-monotonic response to increasing amplitudes of stimulation is due to depolarization block, a change in the membrane potential that prevents the cell from generating action potentials. Main results. We show that the onset for depolarization block depends on the amplitude and frequency of stimulation and reveal the biophysical mechanisms that lead to depolarization block during high frequency stimulation. Our results indicate that differences in transmembrane potassium conductance lead to shifts of the stimulus currents that generate peak spike rates, suggesting that the differential responses of ON and OFF cells may be due to differences in the expression of this current type. We also show that the length of the axon’s high sodium channel band (SOCB) affects non-monotonic responses and the stimulation amplitude that leads to the peak spike rate, suggesting that the length of the SOCB is shorter in ON cells. Significance. This may have important implications for stimulation strategies in visual prostheses.

Real-Time Gene Expression Profiling of Live Shewanella Oneidensis Cells

Energy Technology Data Exchange (ETDEWEB)

Xiaoliang Sunney Xie

2009-03-30

The overall objective of this proposal is to make real-time observations of gene expression in live Shewanella oneidensis cells with high sensitivity and high throughput. Gene expression, a central process to all life, is stochastic because most genes often exist in one or two copies per cell. Although the central dogma of molecular biology has been proven beyond doubt, due to insufficient sensitivity, stochastic protein production has not been visualized in real time in an individual cell at the single-molecule level. We report the first direct observation of single protein molecules as they are generated, one at a time in a single live E. coli cell, yielding quantitative information about gene expression [Science 2006; 311: 1600-1603]. We demonstrated a general strategy for live-cell single-molecule measurements: detection by localization. It is difficult to detect single fluorescence protein molecules inside cytoplasm - their fluorescence is spread by fast diffusion to the entire cell and overwhelmed by the strong autofluorescence. We achieved single-molecule sensitivity by immobilizing the fluorescence protein on the cell membrane, where the diffusion is much slowed. We learned that under the repressed condition protein molecules are produced in bursts, with each burst originating from a stochastically-transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. We also simultaneously published a paper reporting a different method using β-glactosidase as a reporter [Nature 440, 358 (2006)]. Many important proteins are expressed at low levels, inaccessible by previous proteomic techniques. Both papers allowed quantification of protein expression with unprecedented sensitivity and received overwhelming acclaim from the scientific community. The Nature paper has been identified as one of the most-cited papers in the past year [http://esi-topics.com/]. We have also an analytical framework describing the

Live Cells Decreased Methane Production in Intestinal Content of Pigs

Directory of Open Access Journals (Sweden)

Y. L. Gong

2013-06-01

Full Text Available An in vitro gas production technique was used in this study to elucidate the effect of two strains of active live yeast on methane (CH4 production in the large intestinal content of pigs to provide an insight to whether active live yeast could suppress CH4 production in the hindgut of pigs. Treatments used in this study include blank (no substrate and no live yeast cells, control (no live yeast cells and yeast (YST supplementation groups (supplemented with live yeast cells, YST1 or YST2. The yeast cultures contained 1.8×1010 cells per g, which were added at the rates of 0.2 mg and 0.4 mg per ml of the fermented inoculum. Large intestinal contents were collected from 2 Duroc×Landrace×Yorkshire pigs, mixed with a phosphate buffer (1:2, and incubated anaerobically at 39°C for 24 h using 500 mg substrate (dry matter (DM basis. Total gas and CH4 production decreased (p<0.05 with supplementation of yeast. The methane production reduction potential (MRP was calculated by assuming net methane concentration for the control as 100%. The MRP of yeast 2 was more than 25%. Compared with the control group, in vitro DM digestibility (IVDMD and total volatile fatty acids (VFA concentration increased (p<0.05 in 0.4 mg/ml YST1 and 0.2 mg/ml YST2 supplementation groups. Proportion of propionate, butyrate and valerate increased (p<0.05, but that of acetate decreased (p<0.05, which led to a decreased (p<0.05 acetate: propionate (A: P ratio in the both YST2 treatments and the 0.4 mg/ml YST 1 supplementation groups. Hydrogen recovery decreased (p<0.05 with yeast supplementation. Quantity of methanogenic archaea per milliliter of inoculum decreased (p<0.05 with yeast supplementation after 24 h of incubation. Our results suggest that live yeast cells suppressed in vitro CH4 production when inoculated into the large intestinal contents of pigs and shifted the fermentation pattern to favor propionate production together with an increased population of acetogenic

Live-cell imaging: new avenues to investigate retinal regeneration

Directory of Open Access Journals (Sweden)

Manuela Lahne

2017-01-01

Full Text Available Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish (Danio rerio possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.

Effects of high-gradient magnetic fields on living cell machinery

Czech Academy of Sciences Publication Activity Database

Zablotskyy, V.; Lunov, O.; Kubinová, Šárka; Polyakova, T.; Syková, Eva; Dejneka, A.

2016-01-01

Roč. 49, č. 2016 (2016), s. 493003 ISSN 0022-3727 R&D Projects: GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : living cell * magnetic gradient force * cell mechanics * stem cell * magnetic field Subject RIV: FP - Other Medical Disciplines Impact factor: 2.588, year: 2016

Living Polymerization of N -Substituted β-Alanine N -Carboxyanhydrides: Kinetic Investigations and Preparation of an Amphiphilic Block Copoly-β-Peptoid

KAUST Repository

Grossmann, Arlett

2012-07-03

Poly(α-peptoid)s (N-substituted polyglycines) are interesting peptidomimetic biomaterials that have been discussed for many applications. Poly(β-peptoid)s (N-substituted poly-β-alanines), although equally intriguing, have received much less attention. Here we present results that suggest that while N-substituted β-alanine N-carboxyanhydrides can undergo a living nucleophilic ring-opening polymerization, the solubility of poly(β-peptoid)s can be very poor, which contributes to the limited accessibility using other synthetic approaches. The living character of the polymerization was utilized for the preparation of the first polymerized amphiphilic block copoly-β-peptoid. Our results may open a new route towards highly defined functional poly(β-peptoid)s which could represent biomaterials. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous detection of mRNA and protein stem cell markers in live cells

Directory of Open Access Journals (Sweden)

Bao Gang

2009-04-01

Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

Polystyrene-block-Poly(ionic liquid) Copolymers as Work Function Modifiers in Inverted Organic Photovoltaic Cells.

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Park, Jong Baek; Isik, Mehmet; Park, Hea Jung; Jung, In Hwan; Mecerreyes, David; Hwang, Do-Hoon

2018-02-07

Interfacial layers play a critical role in building up the Ohmic contact between electrodes and functional layers in organic photovoltaic (OPV) solar cells. These layers are based on either inorganic oxides (ZnO and TiO 2 ) or water-soluble organic polymers such as poly[(9,9-dioctyl-2,7-fluorene)-alt-(9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7-fluorene)] and polyethylenimine ethoxylated (PEIE). In this work, we have developed a series of novel poly(ionic liquid) nonconjugated block copolymers for improving the performance of inverted OPV cells by using them as work function modifiers of the indium tin oxide (ITO) cathode. Four nonconjugated polyelectrolytes (n-CPEs) based on polystyrene and imidazolium poly(ionic liquid) (PSImCl) were synthesized by reversible addition-fragmentation chain transfer polymerization. The ratio of hydrophobic/hydrophilic block copolymers was varied depending on the ratio of polystyrene to the PSImCl block. The ionic density, which controls the work function of the electrode by forming an interfacial dipole between the electrode and the block copolymers, was easily tuned by simply changing the PSImCl molar ratio. The inverted OPV device with the ITO/PS 29 -b-PSImCl 60 cathode achieved the best power conversion efficiency (PCE) of 7.55% among the synthesized block copolymers, exhibiting an even higher PCE than that of the reference OPV device with PEIE (7.30%). Furthermore, the surface properties of the block copolymers films were investigated by contact angle measurements to explore the influence of the controlled hydrophobic/hydrophilic characters on the device performances.

Autolyse the cell in order to save it? Inducing, then blocking, autolysis as a strategy for delaying cell death in the probiotic Lactobacillus reuteri.

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Zimmerman, Tahl; Gyawali, Rabin; Ibrahim, Salam

2017-10-01

To examine whether choline and its derivatives can be used to preserve viable cells of Lactobacillus reuteri in autolytic models. A phosphate-induced autolytic model in de Man, Rogosa and Sharpe medium (MRS) was used. Viable cell counts were determined by plated on MRS-agar. Choline and hemicholinium-3 (HC-3) significantly blocked autolysis of L. reuteri at 360 mM and 4 mM, respectively. Viable cell counts corroborated these observations. Importantly, autolytically induced cells treated with choline and hemicholinium-3 were significantly more viable then even non-induced cells. Over-production of a known autolytic protein, spirosin, was not attenuated in the presence of choline and hemicholinium-3. Inducing autolysis and then blocking it with choline and its analogs is a promising approach for retaining the viability of L. reuteri cells.

Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer.

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De, Abhijit; Gambhir, Sanjiv Sam

2005-12-01

This study demonstrates a significant advancement of imaging of a distance-dependent physical process, known as the bioluminescent resonance energy transfer (BRET2) signal in living subjects, by using a cooled charge-coupled device (CCD) camera. A CCD camera-based spectral imaging strategy enables simultaneous visualization and quantitation of BRET signal from live cells and cells implanted in living mice. We used the BRET2 system, which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC (DBC) as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. To accomplish this objective in this proof-of-principle study, the donor and acceptor proteins were fused to FKBP12 and FRB, respectively, which are known to interact only in the presence of the small molecule mediator rapamycin. Mammalian cells expressing these fusion constructs were imaged using a cooled-CCD camera either directly from culture dishes or by implanting them into mice. By comparing the emission photon yields in the presence and absence of rapamycin, the specific BRET signal was determined. The CCD imaging approach of BRET signal is particularly appealing due to its capacity to seamlessly bridge the gap between in vitro and in vivo studies. This work validates BRET as a powerful tool for interrogating and observing protein-protein interactions directly at limited depths in living mice.

Mucin2 is Required for Probiotic Agents-Mediated Blocking Effects on Meningitic E. coli-Induced Pathogenicities.

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Yu, Jing-Yi; He, Xiao-Long; Puthiyakunnon, Santhosh; Peng, Liang; Li, Yan; Wu, Li-Sha; Peng, Wen-Ling; Zhang, Ya; Gao, Jie; Zhang, Yao-Yuan; Boddu, Swapna; Long, Min; Cao, Hong; Huang, Sheng-He

2015-10-01

Mucin2 (MUC2), an important regulatory factor in the immune system, plays an important role in the host defense system against bacterial translocation. Probiotics known to regulate MUC2 gene expression have been widely studied, but the interactions among probiotic, pathogens, and mucin gene are still not fully understood. The aim of this study was to investigate the role of MUC2 in blocking effects of probiotics on meningitic E. coli-induced pathogenicities. In this study, live combined probiotic tablets containing living Bifidobacterium, Lactobacillus bulgaricus, and Streptococcus thermophilus were used. MUC2 expression was knocked down in Caco-2 cells by RNA interference. 5-Aza-2'-deoxycytidine (5-Aza-CdR), which enhances mucin-promoted probiotic effects through inducing production of Sadenosyl- L-methionine (SAMe), was used to up-regulate MUC2 expression in Caco-2 cells. The adhesion to and invasion of meningitic E. coli were detected by competition assays. Our studies showed that probiotic agents could block E. coli-caused intestinal colonization, bacteremia, and meningitis in a neonatal sepsis and meningitis rat model. MUC2 gene expression in the neonatal rats given probiotic agents was obviously higher than that of the infected and uninfected control groups without probiotic treatment. The prohibitive effects of probiotic agents on MUC2-knockdown Caco-2 cells infected with E44 were significantly reduced compared with nontransfected Caco-2 cells. Moreover, the results also showed that 5- Aza-CdR, a drug enhancing the production of SAMe that is a protective agent of probiotics, was able to significantly suppress adhesion and invasion of E44 to Caco-2 cells by upregulation of MUC2 expression. Taken together, our data suggest that probiotic agents can efficiently block meningitic E. coli-induced pathogenicities in a manner dependent on MUC2.

[Complete atrioventricular block in Duchenne muscular dystrophy].

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Kuru, Satoshi; Tanahashi, Tamotsu; Matsumoto, Shinjirou; Kitamura, Tetsuya; Konagaya, Masaaki

2012-01-01

We report a case of complete atrioventricular (AV) block in a 40-year-old patient with Duchenne muscular dystrophy (DMD). While he was bed-ridden and required mechanical ventilation, his cardiac involvement was mild. He had the deletion of exon 45-52 in the dystrophin gene. He underwent transient complete AV block and came to require pacemaker implantation due to recurrence of complete AV block ten days after the first attack. Electrophysiological study revealed mild prolonged AH and HV interval. Although DMD patients with AV block have been rarely reported so far, attention should be paid to AV block for patients who prolonged their lives.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

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Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

2014-11-28

Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

International Nuclear Information System (INIS)

Horita, Nobukatsu; Tsuchiya, Kiichiro; Hayashi, Ryohei; Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro; Okamoto, Ryuichi; Nakamura, Tetsuya; Watanabe, Mamoru

2014-01-01

Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

Design of microdevices for long-term live cell imaging

International Nuclear Information System (INIS)

Chen, Huaying; Nordon, Robert E; Rosengarten, Gary; Li, Musen

2012-01-01

Advances in fluorescent live cell imaging provide high-content information that relates a cell's life events to its ancestors. An important requirement to track clonal growth and development is the retention of motile cells derived from an ancestor within the same microscopic field of view for days to weeks, while recording fluorescence images and controlling the mechanical and biochemical microenvironments that regulate cell growth and differentiation. The aim of this study was to design a microwell device for long-term, time-lapse imaging of motile cells with the specific requirements of (a) inoculating devices with an average of one cell per well and (b) retaining progeny of cells within a single microscopic field of view for extended growth periods. A two-layer PDMS microwell culture device consisting of a parallel-plate flow cell bonded on top of a microwell array was developed for cell capture and clonal culture. Cell deposition statistics were related to microwell geometry (plate separation and well depth) and the Reynolds number. Computational fluid dynamics was used to simulate flow in the microdevices as well as cell–fluid interactions. Analysis of the forces acting upon a cell was used to predict cell docking zones, which were confirmed by experimental observations. Cell–fluid dynamic interactions are important considerations for design of microdevices for long-term, live cell imaging. The analysis of force and torque balance provides a reasonable approximation for cell displacement forces. It is computationally less intensive compared to simulation of cell trajectories, and can be applied to a wide range of microdevice geometries to predict the cell docking behavior. (paper)

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

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Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

2014-01-01

Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

Override of the radiation-induced mitotic block in human tumour cells by methylxanthines and its relationship to the potentiation of cytotoxicity

International Nuclear Information System (INIS)

Musk, S.R.R.; Steel, G.G.

1990-01-01

Caffeine, theophylline, theobromine and paraxanthine, were tested for ability to override mitotic block induced by ionizing radiation in the human bladder carcinoma cell line RT112. All were found to partially override the block, at a concentration of 1mM in the order caffeine > theophylline > theobromine = paraxanthine. At a concentration of 1 mM only caffeine was found to potentiate cell killing as well as causing block override; at higher concentrations all had a significant effect on survival but little or no further influence on the degree of block override. It is concluded that override of a mitotic block is not in itself sufficient to cause increased killing when irradiated cells are incubated in the presence of caffeine, and that caffeine exerts its potentiating effect by directly inhibiting repair of damage in DNA or by causing override of radiation-induced inhibition of DNA synthesis. (author)

Override of the radiation-induced mitotic block in human tumour cells by methylxanthines and its relationship to the potentiation of cytotoxicity

Energy Technology Data Exchange (ETDEWEB)

Musk, S.R.R.; Steel, G.G. (Institute of Cancer Research, Sutton (UK). Surrey Branch)

1990-06-01

Caffeine, theophylline, theobromine and paraxanthine, were tested for ability to override mitotic block induced by ionizing radiation in the human bladder carcinoma cell line RT112. All were found to partially override the block, at a concentration of 1mM in the order caffeine > theophylline > theobromine = paraxanthine. At a concentration of 1 mM only caffeine was found to potentiate cell killing as well as causing block override; at higher concentrations all had a significant effect on survival but little or no further influence on the degree of block override. It is concluded that override of a mitotic block is not in itself sufficient to cause increased killing when irradiated cells are incubated in the presence of caffeine, and that caffeine exerts its potentiating effect by directly inhibiting repair of damage in DNA or by causing override of radiation-induced inhibition of DNA synthesis. (author).

Ligament flow during drop-on-demand inkjet printing of bioink containing living cells

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Zhang, Mengyun; Krishnamoorthy, Srikumar; Song, Hongtao; Zhang, Zhengyi; Xu, Changxue

2017-03-01

Organ printing utilizes tissue spheroids or filaments as building blocks to fabricate three-dimensional (3D) functional tissues and organs based on a layer-by-layer manufacturing mechanism. These fabricated tissues and organs are envisioned as alternatives to replace the damaged human tissues and organs, which is emerging as a promising solution to solve the organ donor shortage problem being faced all over the world. Inkjetting, one of the key technologies in organ printing, has been widely developed because of its moderate fabrication cost, good process controllability, and scale-up potentials. There are several key steps towards inkjet-based organ printing: generation of droplets from bioink, fabrication of 3D cellular structures, and post-printing tissue fusion and maturation. The droplet formation process is the first step, affecting the overall feasibility of the envisioned organ printing technology. This paper focuses on the ligament flow of the droplet formation process during inkjet printing of bioink containing living cells and its corresponding effect on post-printing cell viability and cell distribution. It is found that (1) two types of ligament flow are observed: at 30 V (Type I), the ligament flow has two different directions at the locations near the nozzle orifice and the forming droplet; at 60 V (Type II), the ligament flow directions are the same at both locations; (2) compared to Type II, fewer cells are ejected into the primary droplets in Type I, because some cells move back into the nozzle driven by the ligament flow in the positive z direction; and (3) cell viability in both Type I and Type II is around 90% without a significant difference. The resulting knowledge will benefit precise control of printing dynamics during inkjet printing of viscoelastic bioink for 3D biofabrication applications.

A new simple phthalimide-based fluorescent probe for highly selective cysteine and bioimaging for living cells

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Shen, Youming; Zhang, Xiangyang; Zhang, Youyu; Zhang, Chunxiang; Jin, Junling; Li, Haitao

2017-10-01

A new turn-on phthalimide fluorescent probe has designed and synthesized for sensing cysteine (Cys) based on excited state intramolecular proton transfer (ESIPT) process. It is consisted of a 3-hydroxyphthalimide derivative moiety as the fluorophore and an acrylic ester group as a recognition receptor. The acrylic ester acts as an ESIPT blocking agent. Upon addition of cystein, intermolecular nucleophilic attack of cysteine on acrylic ester releases the fluorescent 3-hydroxyphthalimide derivative, thereby enabling the ESIPT process and leading to enhancement of fluorescence. The probe displays high sensitivity, excellent selectivity and with large Stokes shift toward cysteine. The linear interval range of the fluorescence titration ranged from 0 to 1.0 × 10- 5 M and detection limit is low (6 × 10- 8 M). In addition, the probe could be used for bio-imaging in living cells.

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

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Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

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Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

2014-01-01

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

Science.gov (United States)

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

Modulation of radiation-induced apoptosis and G{sub 2}/M block in murine T-lymphoma cells

Energy Technology Data Exchange (ETDEWEB)

Palayoor, S.T.; Macklis, R.M.; Bump, E.A.; Coleman, C.N. [Harvard Medical School, Boston, MA (United States)

1995-03-01

Radiation-induced apoptosis in lymphocyte-derived cell lines is characterized by endonucleolytic cleavage of cellular DNA within hours after radiation exposure. We have studied this phenomenon qualitatively (DNA gel electrophoresis) and quantitatively (diphenylamine reagent assay) in murine EL4 T-lymphoma cells exposed to {sup 137}Cs {gamma} irradiation. Fragmentation was discernible within 18-24 h after exposure. It increased with time and dose and reached a plateau after 8 Gy of {gamma} radiation. We studied the effect of several pharmacological agents on the radiation-induced G{sub 2}/M block and DNA fragmentation. The agents which reduced the radiation-induced G{sub 2}/M-phase arrest (caffeine, theobromine, theophylline and 2-aminopurine) enhanced the degree of DNA fragmentation at 24 h. In contrast, the agents which sustained the radiation-induced G{sub 2}/M-phase arrest (TPA, DBcAMP, IBMX and 3-aminobenzamide) inhibited the DNA fragmentation at 24 h. These studies on EL4 lymphoma cells are consistent with the hypothesis that cells with radiation-induced genetic damage are eliminated by apoptosis subsequent to a G{sub 2}/M block. Furthermore, it may be possible to modulate the process of radiation-induced apoptosis in lymphoma cells with pharmacological agents that modify the radiation-induced G{sub 2}/M block, and to use this effect in the treatment of patients with malignant disease. 59 refs., 7 figs.

Direct Visualization of De novo Lipogenesis in Single Living Cells

Science.gov (United States)

Li, Junjie; Cheng, Ji-Xin

2014-10-01

Increased de novo lipogenesis is being increasingly recognized as a hallmark of cancer. Despite recent advances in fluorescence microscopy, autoradiography and mass spectrometry, direct observation of de novo lipogenesis in living systems remains to be challenging. Here, by coupling stimulated Raman scattering (SRS) microscopy with isotope labeled glucose, we were able to trace the dynamic metabolism of glucose in single living cells with high spatial-temporal resolution. As the first direct visualization, we observed that glucose was largely utilized for lipid synthesis in pancreatic cancer cells, which occurs at a much lower rate in immortalized normal pancreatic epithelial cells. By inhibition of glycolysis and fatty acid synthase (FAS), the key enzyme for fatty acid synthesis, we confirmed the deuterium labeled lipids in cancer cells were from de novo lipid synthesis. Interestingly, we also found that prostate cancer cells exhibit relatively lower level of de novo lipogenesis, but higher fatty acid uptake compared to pancreatic cancer cells. Together, our results demonstrate a valuable tool to study dynamic lipid metabolism in cancer and other disorders.

Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors

Directory of Open Access Journals (Sweden)

Yuliang Liu

2011-01-01

Full Text Available Virus-host interactions play key roles in promoting efficient egress of many RNA viruses, including Ebola virus (EBOV or “e” and Marburg virus (MARV or “m”. Late- (L- domains conserved in viral matrix proteins recruit specific host proteins, such as Tsg101 and Nedd4, to facilitate the budding process. These interactions serve as attractive targets for the development of broad-spectrum budding inhibitors. A major gap still exists in our understanding of the mechanism of filovirus budding due to the difficulty in detecting virus-host complexes and mapping their trafficking patterns in the natural environment of the cell. To address this gap, we used a bimolecular complementation (BiMC approach to detect, localize, and follow the trafficking patterns of eVP40-Tsg101 complexes in live mammalian cells. In addition, we used the BiMC approach along with a VLP budding assay to test small molecule inhibitors identified by in silico screening for their ability to block eVP40 PTAP-mediated interactions with Tsg101 and subsequent budding of eVP40 VLPs. We demonstrated the potential broad spectrum activity of a lead candidate inhibitor by demonstrating its ability to block PTAP-dependent binding of HIV-1 Gag to Tsg101 and subsequent egress of HIV-1 Gag VLPs.

Perturbed cholesterol and vesicular trafficking associated with dengue blocking in Wolbachia-infected Aedes aegypti cells.

Science.gov (United States)

Geoghegan, Vincent; Stainton, Kirsty; Rainey, Stephanie M; Ant, Thomas H; Dowle, Adam A; Larson, Tony; Hester, Svenja; Charles, Philip D; Thomas, Benjamin; Sinkins, Steven P

2017-09-13

Wolbachia are intracellular maternally inherited bacteria that can spread through insect populations and block virus transmission by mosquitoes, providing an important approach to dengue control. To better understand the mechanisms of virus inhibition, we here perform proteomic quantification of the effects of Wolbachia in Aedes aegypti mosquito cells and midgut. Perturbations are observed in vesicular trafficking, lipid metabolism and in the endoplasmic reticulum that could impact viral entry and replication. Wolbachia-infected cells display a differential cholesterol profile, including elevated levels of esterified cholesterol, that is consistent with perturbed intracellular cholesterol trafficking. Cyclodextrins have been shown to reverse lipid accumulation defects in cells with disrupted cholesterol homeostasis. Treatment of Wolbachia-infected Ae. aegypti cells with 2-hydroxypropyl-β-cyclodextrin restores dengue replication in Wolbachia-carrying cells, suggesting dengue is inhibited in Wolbachia-infected cells by localised cholesterol accumulation. These results demonstrate parallels between the cellular Wolbachia viral inhibition phenotype and lipid storage genetic disorders. Wolbachia infection of mosquitoes can block dengue virus infection and is tested in field trials, but the mechanism of action is unclear. Using proteomics, Geoghegan et al. here identify effects of Wolbachia on cholesterol homeostasis and dengue virus replication in Aedes aegypti.

Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.

Directory of Open Access Journals (Sweden)

Jan M Bruder

Full Text Available Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1 localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin's physiological response to UV radiation.

Quantification of GPCR internalization by single-molecule microscopy in living cells.

NARCIS (Netherlands)

Serge, A.; Keijzer, S. de; Hemert, F. Van; Hickman, M.R.; Hereld, D.; Spaink, H.P.; Schmidt, T.; Snaar-Jagalska, B.E.

2011-01-01

Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells.

Transverse mechanical properties of cell walls of single living plant cells probed by laser-generated acoustic waves.

Science.gov (United States)

Gadalla, Atef; Dehoux, Thomas; Audoin, Bertrand

2014-05-01

Probing the mechanical properties of plant cell wall is crucial to understand tissue dynamics. However, the exact symmetry of the mechanical properties of this anisotropic fiber-reinforced composite remains uncertain. For this reason, biologically relevant measurements of the stiffness coefficients on individual living cells are a challenge. For this purpose, we have developed the single-cell optoacoustic nanoprobe (SCOPE) technique, which uses laser-generated acoustic waves to probe the stiffness, thickness and viscosity of live single-cell subcompartments. This all-optical technique offers a sub-micrometer lateral resolution, nanometer in-depth resolution, and allows the non-contact measurement of the mechanical properties of live turgid tissues without any assumption of mechanical symmetry. SCOPE experiments reveal that single-cell wall transverse stiffness in the direction perpendicular to the epidermis layer of onion cells is close to that of cellulose. This observation demonstrates that cellulose microfibrils are the main load-bearing structure in this direction, and suggests strong bonding of microfibrils by hemicelluloses. Altogether our measurement of the viscosity at high frequencies suggests that the rheology of the wall is dominated by glass-like dynamics. From a comparison with literature, we attribute this behavior to the influence of the pectin matrix. SCOPE's ability to unravel cell rheology and cell anisotropy defines a new class of experiments to enlighten cell nano-mechanics.

Live-cell super-resolution imaging of intrinsically fast moving flagellates

Science.gov (United States)

Glogger, M.; Stichler, S.; Subota, I.; Bertlein, S.; Spindler, M.-C.; Teßmar, J.; Groll, J.; Engstler, M.; Fenz, S. F.

2017-02-01

Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μs. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. , which features invited work from the best early-career researchers working within the scope of J Phys D. This project is part of the Journal of Physics series’ 50th anniversary celebrations in 2017. Susanne Fenz was selected by the Editorial Board of J Phys D as an Emerging Talent/Leader.

Axial tomography in live cell laser microscopy

Science.gov (United States)

Richter, Verena; Bruns, Sarah; Bruns, Thomas; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

2017-09-01

Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.

Tracking chemical changes in a live cell: Biomedical applications of SR-FTIR spectromicroscopy

International Nuclear Information System (INIS)

Holman, Hoi-Ying N.; Martin, Michael C.; McKinney, Wayne R.

2002-01-01

Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging bioanalytical and imaging tool. This unique technique provides mid-infrared (IR) spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Thus it enables researchers to locate, identify, and track specific chemical events within an individual living mammalian cell. Mid-IR photons are too low in energy (0.05 - 0.5 eV) to either break bonds or to cause ionization. In this review, we show that the synchrotron IR beam has no detectable effects on the short- and long-term viability, reproductive integrity, cell-cycle progression, and mitochondrial metabolism in living human cells, and produces only minimal sample heating (< 0.5 degrees C). We will then present several examples demonstrating the application potentials of SR-FTIR spectromicroscopy in biomedical research. These will include monitoring living cells progressing through the cell cycle, including death, and cells reacting to dilute concentrations of toxins

Tracking chemical changes in a live cell: Biomedical applications of SR-FTIR spectromicroscopy

Energy Technology Data Exchange (ETDEWEB)

Holman, Hoi-Ying N.; Martin, Michael C.; McKinney, Wayne R.

2002-07-25

Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging bioanalytical and imaging tool. This unique technique provides mid-infrared (IR) spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Thus it enables researchers to locate, identify, and track specific chemical events within an individual living mammalian cell. Mid-IR photons are too low in energy (0.05 - 0.5 eV) to either break bonds or to cause ionization. In this review, we show that the synchrotron IR beam has no detectable effects on the short- and long-term viability, reproductive integrity, cell-cycle progression, and mitochondrial metabolism in living human cells, and produces only minimal sample heating (< 0.5 degrees C). We will then present several examples demonstrating the application potentials of SR-FTIR spectromicroscopy in biomedical research. These will include monitoring living cells progressing through the cell cycle, including death, and cells reacting to dilute concentrations of toxins.

In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

International Nuclear Information System (INIS)

Ribot, E.J.; Foster, P.J.

2012-01-01

The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. (orig.)

In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

Energy Technology Data Exchange (ETDEWEB)

Ribot, E.J. [University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, ON (Canada); Foster, P.J. [University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, ON (Canada); University of Western Ontario, Department of Medical Biophysics, London, ON (Canada)

2012-09-15

The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. (orig.)

Well-defined block copolymers for gene delivery to dendritic cells: probing the effect of polycation chain-length.

Science.gov (United States)

Tang, Rupei; Palumbo, R Noelle; Nagarajan, Lakshmi; Krogstad, Emily; Wang, Chun

2010-03-03

The development of safe and efficient polymer carriers for DNA vaccine delivery requires mechanistic understanding of structure-function relationship of the polymer carriers and their interaction with antigen-presenting cells. Here we have synthesized a series of diblock copolymers with well-defined chain-length using atom transfer radical polymerization and characterized the influence of polycation chain-length on the physico-chemical properties of the polymer/DNA complexes as well as the interaction with dendritic cells. The copolymers consist of a hydrophilic poly(ethylene glycol) block and a cationic poly(aminoethyl methacrylate) (PAEM) block. The average degree of polymerization (DP) of the PAEM block was varied among 19, 39, and 75, with nearly uniform distribution. With increasing PAEM chain-length, polyplexes formed by the diblock copolymers and plasmid DNA had smaller average particle size and showed higher stability against electrostatic destabilization by salt and heparin. The polymers were not toxic to mouse dendritic cells (DCs) and only displayed chain-length-dependent toxicity at a high concentration (1mg/mL). In vitro gene transfection efficiency and polyplex uptake in DCs were also found to correlate with chain-length of the PAEM block with the longer polymer chain favoring transfection and cellular uptake. The polyplexes induced a modest up-regulation of surface markers for DC maturation that was not significantly dependent on PAEM chain-length. Finally, the polyplex prepared from the longest PAEM block (DP of 75) achieved an average of 20% enhancement over non-condensed anionic dextran in terms of uptake by DCs in the draining lymph nodes 24h after subcutaneous injection into mice. Insights gained from studying such structurally well-defined polymer carriers and their interaction with dendritic cells may contribute to improved design of practically useful DNA vaccine delivery systems. Copyright 2009 Elsevier B.V. All rights reserved.

Radiation block of bone marrow cell mitoses and the effect of insulin

Energy Technology Data Exchange (ETDEWEB)

Barkalaya, A I

1976-01-01

Insulin (0.15 - 0.2 units/kg) has been administered to white rats immediately after the exposure to 750 R, at the background of hypercorticoidism. This resulted in the inhibition of the development of the post-irradiation-stress-hyperglycemia; and the mitotic index of the bone marrow cells at the time of the mitosis block was higher than in the control irradiated rats. Insulin administration at the peak of radiation sickness during hypercorticoidism levelled hyperglycemia, stimulated the mitotic activity of cells of the bone marrow and the regeneration of the latter.

Effects of high-gradient magnetic fields on living cell machinery

Czech Academy of Sciences Publication Activity Database

Zablotskyy, Vitaliy A.; Lunov, Oleg; Kubinová, Šárka; Polyakova, Tetyana; Syková, E.; Dejneka, Alexandr

2016-01-01

Roč. 49, č. 49 (2016), s. 1-23, č. článku 493003. ISSN 0022-3727 R&D Projects: GA MŠk LO1409 Grant - others:FUNBIO(XE) CZ.2.16/3.1.00/21568 Institutional support: RVO:68378271 Keywords : living cell * magnetic gradient force * cell mechanics * stem cell * magnetic field Subject RIV: BO - Biophysics Impact factor: 2.588, year: 2016

Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C.

Science.gov (United States)

Maass, Philipp G; Barutcu, A Rasim; Weiner, Catherine L; Rinn, John L

2018-03-15

Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs. Copyright © 2018 Elsevier Inc. All rights reserved.

The lived experiences of adolescents with sickle cell disease in Kingston, Jamaica

Directory of Open Access Journals (Sweden)

Andrea Brown Forrester

2015-09-01

Full Text Available Aim: To explore the lived experiences of adolescents with sickle cell disease, in Kingston, Jamaica. Method: A descriptive qualitative design was used for this research. In-depth interviews were conducted with six adolescents with sickle cell disease at a Sickle Cell Unit operated by the University of the West Indies. Interviews were audiotaped, transcribed, and thematically analyzed. Results: The majority of the adolescents demonstrated a positive self-concept. They reported strong family, school, and peer support which made them feel accepted. All were actively engaged in social activities such as parties, but had challenges participating in sporting activities. Various coping strategies were utilized to address challenges of the disease including praying, watching television, and surfing the Internet. Conclusion: Sickle cell disease can be very challenging for the adolescent, but with positive self-concept and increased social support, especially from family and peers, these adolescents were able to effectively cope with their condition and live productive lives.

The lived experiences of adolescents with sickle cell disease in Kingston, Jamaica.

Science.gov (United States)

Forrester, Andrea Brown; Barton-Gooden, Antoinette; Pitter, Cynthia; Lindo, Jascinth L M

2015-01-01

To explore the lived experiences of adolescents with sickle cell disease, in Kingston, Jamaica. A descriptive qualitative design was used for this research. In-depth interviews were conducted with six adolescents with sickle cell disease at a Sickle Cell Unit operated by the University of the West Indies. Interviews were audiotaped, transcribed, and thematically analyzed. The majority of the adolescents demonstrated a positive self-concept. They reported strong family, school, and peer support which made them feel accepted. All were actively engaged in social activities such as parties, but had challenges participating in sporting activities. Various coping strategies were utilized to address challenges of the disease including praying, watching television, and surfing the Internet. Sickle cell disease can be very challenging for the adolescent, but with positive self-concept and increased social support, especially from family and peers, these adolescents were able to effectively cope with their condition and live productive lives.

Live cell imaging combined with high-energy single-ion microbeam

Science.gov (United States)

Guo, Na; Du, Guanghua; Liu, Wenjing; Guo, Jinlong; Wu, Ruqun; Chen, Hao; Wei, Junzhe

2016-03-01

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10-3 s-1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10-2 s-1.

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

International Nuclear Information System (INIS)

Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille; Rochel, Natacha; Mély, Yves; Didier, Pascal; Weiss, Etienne

2013-01-01

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

Energy Technology Data Exchange (ETDEWEB)

Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France); Rochel, Natacha [Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, CNRS/INSERM/Université de Strasbourg, rue Laurent Fries, 67404 Illkirch (France); Mély, Yves; Didier, Pascal [Faculté de Pharmacie, UMR 7213, CNRS/Université de Strasbourg, route du Rhin, 67401 Illkirch (France); Weiss, Etienne, E-mail: eweiss@unistra.fr [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France)

2013-04-01

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.

Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

Science.gov (United States)

Vaccari, L; Birarda, G; Businaro, L; Pacor, S; Grenci, G

2012-06-05

Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

Rare Coumarins Induce Apoptosis, G1 Cell Block and Reduce RNA Content in HL60 Cells

Directory of Open Access Journals (Sweden)

Widelski Jarosław

2017-02-01

Full Text Available The rare coumarins stenocarpin, stenocarpin isobutyrate, oficinalin, oficinalin isobutyrate, 8-methoxypeucedanin and the known xanthotoxin, isoimperatorin, bergapten, peucedanin and 8–methoxyisoimperatorin were isolated from Peucedanum luxurians Tamamsch. (Apiaceae and identified by means of spectral data (1D and 2D NMR. Their immunomodulating activity was evaluated by flow cytometry and their influence on HL60 cells as well as on PHA-stimulated PBLs was tested. All tested coumarins induce apoptosis (maximal in the 48 h culture and decrease cell proliferation in a time- and dose-dependent manner, especially in HL60 cells. They also induce partial G1 block, but only in HL60 cells (at 100 µM concentrations. Dose-dependent reduction of RNA content was also found in G1 cells treated by the coumarins. All of the tested coumarins also possessed immunomodulatory activities. Bergapten and xanthotoxin were found to be the best candidates for further evaluation as anti-cancer drugs.

Label-free evanescent microscopy for membrane nano-tomography in living cells.

Science.gov (United States)

Bon, Pierre; Barroca, Thomas; Lévèque-Fort, Sandrine; Fort, Emmanuel

2014-11-01

We show that through-the-objective evanescent microscopy (epi-EM) is a powerful technique to image membranes in living cells. Readily implementable on a standard inverted microscope, this technique enables full-field and real-time tracking of membrane processes without labeling and thus signal fading. In addition, we demonstrate that the membrane/interface distance can be retrieved with 10 nm precision using a multilayer Fresnel model. We apply this nano-axial tomography of living cell membranes to retrieve quantitative information on membrane invagination dynamics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.

Science.gov (United States)

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Moazzeni, Mohammad; Soheili, Zahra Soheila; Samiee, Shahram

2010-12-01

In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.

Model system for plant cell biology: GFP imaging in living onion epidermal cells

Science.gov (United States)

Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

1999-01-01

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

Living labeling techniques of mesenchymal stem cells

International Nuclear Information System (INIS)

Dong Qingyu; Chen Li

2007-01-01

Mesenchymal stem cells (MSCs) are well known for their self-renew and multi- differentiation potentiality. With the transplantation of the MSCs which can promote the regeneration and repair of the injured tissue, a new route for the treatment of dieases is hopeful to be effective. To trace the distribution, migration, proliferation and differentiation of the implanted MSCs, there need effective labeling techniques, especially living labeling techniques. (authors)

Digital Holographic Microscopy: Quantitative Phase Imaging and Applications in Live Cell Analysis

Science.gov (United States)

Kemper, Björn; Langehanenberg, Patrik; Kosmeier, Sebastian; Schlichthaber, Frank; Remmersmann, Christian; von Bally, Gert; Rommel, Christina; Dierker, Christian; Schnekenburger, Jürgen

The analysis of complex processes in living cells creates a high demand for fast and label-free methods for online monitoring. Widely used fluorescence methods require specific labeling and are often restricted to chemically fixated samples. Thus, methods that offer label-free and minimally invasive detection of live cell processes and cell state alterations are of particular interest. In combination with light microscopy, digital holography provides label-free, multi-focus quantitative phase imaging of living cells. In overview, several methods for digital holographic microscopy (DHM) are presented. First, different experimental setups for the recording of digital holograms and the modular integration of DHM into common microscopes are described. Then the numerical processing of digitally captured holograms is explained. This includes the description of spatial and temporal phase shifting techniques, spatial filtering based reconstruction, holographic autofocusing, and the evaluation of self-interference holograms. Furthermore, the usage of partial coherent light and multi-wavelength approaches is discussed. Finally, potentials of digital holographic microscopy for quantitative cell imaging are illustrated by results from selected applications. It is shown that DHM can be used for automated tracking of migrating cells and cell thickness monitoring as well as for refractive index determination of cells and particles. Moreover, the use of DHM for label-free analysis in fluidics and micro-injection monitoring is demonstrated. The results show that DHM is a highly relevant method that allows novel insights in dynamic cell biology, with applications in cancer research and for drugs and toxicity testing.

Molecular beacon nanosensors for live cell detection and tracking differentiation and reprogramming

DEFF Research Database (Denmark)

Ilieva, Mirolyuba

2013-01-01

open to closed state within living cells. Using MBs targeting pluripotent stem cell markers we demonstrated reverse into a more immature state of LUHMES induced by neurosphere-like growth conditions. Moreover, we have been able to trace localisation of this particular population during differentiation...... in separation of fluorophore from quencher and thereby emission of a fluorescent signal that can be detected. In this project the usability and applicability of MBs for live cell detection and tracing of gene expression was demonstrated. MBs library targeting gene markers for pluripotent stem cells as well...... and thus demonstrate the usability of MBs for monitoring cell behaviour within 3D clusters. Finally, MBs detection of expression of human pluripotent markers after reprograming of adult somatic cells with plasmid codding for mouse transcription factors was demonstrated. In conclusion, the method of using...

Retention of CXCR4 in the endoplasmic reticulum blocks dissemination of a T cell hybridoma.

Science.gov (United States)

Zeelenberg, I S; Ruuls-Van Stalle, L; Roos, E

2001-07-01

The dissemination of T cell hybridomas to multiple nonhematopoietic tissues is blocked by pertussis toxin, suggesting the involvement of a chemokine. To study whether this chemokine is SDF-1, we employed a strategy proposed previously for gene therapy of AIDS, whereby the SDF-1 receptor CXCR4 (also a coreceptor for HIV) is retained in the endoplasmic reticulum (ER) and fails to reach the cell surface. We transfected SDF-1, carrying an ER retention sequence, into a T cell hybridoma. This altered chemokine is retained in the ER, where it binds CXCR4 and prevents the latter protein from reaching the surface. These cells failed to migrate toward SDF-1 or to invade fibroblast monolayers, although they could still migrate toward thymus and activation-regulated chemokine (TARC) and invade TARC-treated monolayers. Furthermore, the ability of the transfected cells to disseminate to multiple organs upon intravenous injection into mice was abolished. This dissemination reflects the in vivo migration patterns of activated and memory T cells into nonhematopoietic tissues, which is thus likely to depend on CXCR4. Attempts to block CXCR4 function as a therapy for AIDS may affect this migration with consequences for T cell function. Our results also suggest a decisive role for CXCR4 in the dissemination of hematopoietic malignancies expressing this receptor.

[Non-invasive analysis of proteins in living cells using NMR spectroscopy].

Science.gov (United States)

Tochio, Hidehito; Murayama, Shuhei; Inomata, Kohsuke; Morimoto, Daichi; Ohno, Ayako; Shirakawa, Masahiro

2015-01-01

NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest "in-cell NMR" approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR. The cells are grown in an NMR active isotope-enriched medium to ensure that the overexpressed proteins are labeled with the stable isotopes. Thus the obtained NMR spectra, which are derived from labeled proteins, contain atomic-level information about the structure and dynamics of the proteins. Recent progress enables us to work with higher eukaryotic cells such as HeLa and HEK293 cells, for which a number of techniques have been developed to achieve isotope labeling of the specific target protein. In this review, we describe successful use of electroporation for in-cell NMR. In addition, (19)F-NMR to characterize protein-ligand interactions in cells is presented. Because (19)F nuclei rarely exist in natural cells, when (19)F-labeled proteins are delivered into cells and (19)F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.

Detecting infrared luminescence and non-chemical signaling of living cells: single cell mid-IR spectroscopy in cryogenic environments

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Pereverzev, Sergey

2017-02-01

Many life-relevant interaction energies are in IR range, and it is reasonable to believe that some biochemical reactions inside cells can results in emission of IR photons. Cells can use this emission for non-chemical and non-electrical signaling. Detecting weak infrared radiation from live cells is complicated because of strong thermal radiation background and absorption of radiation by tissues. A microfluidic device with live cells inside a vacuum cryogenic environment should suppress this background, and thereby permit observation of live cell auto-luminescence or signaling in the IR regime. One can make IR-transparent windows not emitting in this range, so only the cell and a small amount of liquid around it will emit infrared radiation. Currently mid-IR spectroscopy of single cells requires the use of a synchrotron source to measure absorption or reflection spectra. Decreasing of thermal radiation background will allow absorption and reflection spectroscopy of cells without using synchrotron light. Moreover, cell auto-luminescence can be directly measured. The complete absence of thermal background radiation for cryogenically cooled samples allows the use IR photon-sensitive detectors and obtaining single molecule sensitivity in IR photo-luminescence measurements. Due to low photon energies, photo-luminescence measurements will be non-distractive for pressures samples. The technique described here is based upon US patent 9366574.

Atomic force microscopy as a tool for the investigation of living cells.

Science.gov (United States)

Morkvėnaitė-Vilkončienė, Inga; Ramanavičienė, Almira; Ramanavičius, Arūnas

2013-01-01

Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.

Block copolymer directed synthesis of mesoporous TiO 2 for dye-sensitized solar cells

KAUST Repository

Nedelcu, Mihaela; Lee, Jinwoo; Crossland, Edward J. W.; Warren, Scott C.; Orilall, M. Christopher; Guldin, Stefan; Hü ttner, Sven; Ducati, Catarina; Eder, Dominik; Wiesner, Ulrich; Steiner, Ullrich; Snaith, Henry J.

2009-01-01

The morphology of TiO2 plays an important role in the operation of solid-state dye-sensitized solar cells. By using polyisoprene-block- ethyleneoxide (PI-b-PEO) copolymers as structure directing agents for a sol-gel based synthesis of mesoporous TiO

Live cell imaging combined with high-energy single-ion microbeam

Energy Technology Data Exchange (ETDEWEB)

Guo, Na; Du, Guanghua, E-mail: gh-du@impcas.ac.cn; Liu, Wenjing; Wu, Ruqun; Wei, Junzhe [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Guo, Jinlong [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Northwest Normal University, Lanzhou (China); Chen, Hao [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Institute of Nuclear Science and Technology, University of Lanzhou, Lanzhou (China)

2016-03-15

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10{sup −3} s{sup −1} and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10{sup −2} s{sup −1}.

Live cell imaging combined with high-energy single-ion microbeam

International Nuclear Information System (INIS)

Guo, Na; Du, Guanghua; Liu, Wenjing; Wu, Ruqun; Wei, Junzhe; Guo, Jinlong; Chen, Hao

2016-01-01

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10"−"3 s"−"1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10"−"2 s"−"1.

Living biointerfaces based on non-pathogenic bacteria support stem cell differentiation

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Hay, Jake J.; Rodrigo-Navarro, Aleixandre; Hassi, Karoliina; Moulisova, Vladimira; Dalby, Matthew J.; Salmeron-Sanchez, Manuel

2016-02-01

Lactococcus lactis, a non-pathogenic bacteria, has been genetically engineered to express the III7-10 fragment of human fibronectin as a membrane protein. The engineered L. lactis is able to develop biofilms on different surfaces (such as glass and synthetic polymers) and serves as a long-term substrate for mammalian cell culture, specifically human mesenchymal stem cells (hMSC). This system constitutes a living interface between biomaterials and stem cells. The engineered biofilms remain stable and viable for up to 28 days while the expressed fibronectin fragment induces hMSC adhesion. We have optimised conditions to allow long-term mammalian cell culture, and found that the biofilm is functionally equivalent to a fibronectin-coated surface in terms of osteoblastic differentiation using bone morphogenetic protein 2 (BMP-2) added to the medium. This living bacteria interface holds promise as a dynamic substrate for stem cell differentiation that can be further engineered to express other biochemical cues to control hMSC differentiation.

Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells.

Science.gov (United States)

Schudt, Gordian; Dolnik, Olga; Kolesnikova, Larissa; Biedenkopf, Nadine; Herwig, Astrid; Becker, Stephan

2015-10-01

Transport of ebolavirus (EBOV) nucleocapsids from perinuclear viral inclusions, where they are formed, to the site of budding at the plasma membrane represents an obligatory step of virus assembly. Until now, no live-cell studies on EBOV nucleocapsid transport have been performed, and participation of host cellular factors in this process, as well as the trajectories and speed of nucleocapsid transport, remain unknown. Live-cell imaging of EBOV-infected cells treated with different inhibitors of cellular cytoskeleton was used for the identification of cellular proteins involved in the nucleocapsid transport. EBOV nucleocapsids were visualized by expression of green fluorescent protein (GFP)-labeled nucleocapsid viral protein 30 (VP30) in EBOV-infected cells. Incorporation of the fusion protein VP30-GFP into EBOV nucleocapsids was confirmed by Western blot and indirect immunofluorescence analyses. Importantly, VP30-GFP fluorescence was readily detectable in the densely packed nucleocapsids inside perinuclear viral inclusions and in the dispersed rod-like nucleocapsids located outside of viral inclusions. Live-cell imaging of EBOV-infected cells revealed exit of single nucleocapsids from the viral inclusions and their intricate transport within the cytoplasm before budding at the plasma membrane. Nucleocapsid transport was arrested upon depolymerization of actin filaments (F-actin) and inhibition of the actin-nucleating Arp2/3 complex, and it was not altered upon depolymerization of microtubules or inhibition of N-WASP. Actin comet tails were often detected at the rear end of nucleocapsids. Marginally located nucleocapsids entered filopodia, moved inside, and budded from the tip of these thin cellular protrusions. Live-cell imaging of EBOV-infected cells revealed actin-dependent long-distance transport of EBOV nucleocapsids before budding at the cell surface. These findings provide useful insights into EBOV assembly and have potential application in the development

Fourier-transform infrared spectroscopy for rapid screening and live-cell monitoring: application to nanotoxicology.

Science.gov (United States)

Sundaram, S K; Sacksteder, Colette A; Weber, Thomas J; Riley, Brian J; Addleman, R Shane; Harrer, Bruce J; Peterman, John W

2013-01-01

A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live cells interact with an external stimulus, such as a nanosized particle, and the toxicity and broad risk associated with these stimuli. It is difficult to capture the complexity and dynamics of these interactions by following omics-based approaches exclusively, which can be expensive and time-consuming. Attenuated total reflectance-Fourier transform infrared spectroscopy is well suited to provide noninvasive live-cell monitoring of cellular responses to potentially toxic nanosized particles or other stimuli. This alternative approach provides the ability to carry out rapid toxicity screenings and nondisruptive monitoring of live-cell cultures. We review the technical basis of the approach, the instrument configuration and interface with the biological media, the various effects that impact the data, subsequent data analysis and toxicity, and present some preliminary results on live-cell monitoring.

[Methods of substances and organelles introduction in living cell for cell engineering technologies].

Science.gov (United States)

Nikitin, V A

2007-01-01

We have presented the classification of more than 40 methods of genetic material, substances and organelles introduction into a living cell. Each of them has its characteristic advantages, disadvantages and limitations with respect to cell viability, transfer efficiency, general applicability, and technical requirements. It this article we have enlarged on the description of our developments of several new and improved approaches, methods and devices of the direct microinjection into a single cell and cell microsurgery with the help of glass micropipettes. The problem of low efficiency of mammalian cloning is discussed with emphasis on the necessity of expertizing of each step of single cell reconstruction to begin with microsurgical manipulations and necessity of the development of such methods of single cell resonstruction that could minimize the possible damage of the cell.

Omeprazole blocks STAT6 binding to the eotaxin-3 promoter in eosinophilic esophagitis cells.

Directory of Open Access Journals (Sweden)

Xi Zhang

Full Text Available Patients who have esophageal eosinophilia without gastroesophageal reflux disease (GERD nevertheless can respond to proton pump inhibitors (PPIs, which can have anti-inflammatory actions independent of effects on gastric acid secretion. In esophageal cell cultures, omeprazole has been reported to inhibit Th2 cytokine-stimulated expression of eotaxin-3, an eosinophil chemoattractant contributing to esophageal eosinophilia in eosinophilic esophagitis (EoE. The objective of this study was to elucidate molecular mechanisms underlying PPI inhibition of IL-4-stimulated eotaxin-3 production by esophageal cells.Telomerase-immortalized and primary cultures of esophageal squamous cells from EoE patients were treated with IL-4 in the presence or absence of acid-activated omeprazole or lansoprazole. We measured eotaxin-3 protein secretion by ELISA, mRNA expression by PCR, STAT6 phosphorylation and nuclear translocation by Western blotting, eotaxin-3 promoter activation by an exogenous reporter construct, and STAT6, RNA polymerase II, and trimethylated H3K4 binding to the endogenous eotaxin-3 promoter by ChIP assay. Omeprazole in concentrations ≥5 µM significantly decreased IL-4-stimulated eotaxin-3 protein secretion and mRNA expression. Lansoprazole also blocked eotaxin-3 protein secretion. Omeprazole had no effect on eotaxin-3 mRNA stability or on STAT6 phosphorylation and STAT6 nuclear translocation. Rather, omeprazole blocked binding of IL-4-stimulated STAT6, RNA polymerase II, and trimethylated H3K4 to the eotaxin-3 promoter.PPIs, in concentrations achieved in blood with conventional dosing, significantly inhibit IL-4-stimulated eotaxin-3 expression in EoE esophageal cells and block STAT6 binding to the promoter. These findings elucidate molecular mechanisms whereby patients with Th2 cytokine-driven esophageal eosinophilia can respond to PPIs, independent of effects on gastric acid secretion.

Neutron scattering to study membrane systems: from lipid vesicles to living cells.

Energy Technology Data Exchange (ETDEWEB)

Nickels, Jonathan D. [ORNL; Chatterjee, Sneha [ORNL; Stanley, Christopher B. [ORNL; Qian, Shuo [ORNL; Cheng, Xiaolin [ORNL; Myles, Dean A A [ORNL; Standaert, Robert F. [ORNL; Elkins, James G. [ORNL; Katsaras, John [ORNL

2017-03-01

The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent work using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.

Ultrasound-Guided Phrenic Nerve Block for Intractable Hiccups following Placement of Esophageal Stent for Esophageal Squamous Cell Carcinoma.

Science.gov (United States)

Arsanious, David; Khoury, Spiro; Martinez, Edgar; Nawras, Ali; Filatoff, Gregory; Ajabnoor, Hossam; Darr, Umar; Atallah, Joseph

2016-05-01

Hiccups are actions consisting of sudden contractions of the diaphragm and intercostals followed by a sudden inspiration and transient closure of the vocal cords. They are generally short lived and benign; however, in extreme and rare cases, such as esophageal carcinoma, they can become persistent or intractable, up to and involving significant pain, dramatically impacting the patient's quality of life. This case involves a 60-year-old man with a known history of squamous cell carcinoma of the esophagus. He was considered to have high surgical risk, and therefore he received palliative care through the use of fully covered metallic esophageal self-expandable stents due to a spontaneous perforated esophagus, after which he developed intractable hiccups and associated mediastinal pain. Conservative treatment, including baclofen, chlorpromazine, metoclopramide, and omeprazole, provided no relief for his symptoms. The patient was referred to pain management from gastroenterology for consultation on pain control. He ultimately received an ultrasound-guided left phrenic nerve block with bupivacaine and depomedrol, and 3 days later underwent the identical procedure on the right phrenic nerve. This led to complete resolution of his hiccups and associated mediastinal pain. At follow-up, 2 and 4 weeks after the left phrenic nerve block, the patient was found to maintain complete alleviation of the hiccups. Esophageal dilatation and/or phrenic or vagal afferent fiber irritation can be suspected in cases of intractable hiccups secondary to esophageal stenting. Regional anesthesia of the phrenic nerve through ultrasound guidance offers a long-term therapeutic option for intractable hiccups and associated mediastinal pain in selected patients with esophageal carcinoma after stent placement. Esophageal stent, esophageal stenting, intractable hiccups, intractable singultus, phrenic nerve block, phrenic nerve, ultrasound, palliative care, esophageal carcinoma.

Thermodynamics of protein destabilization in live cells.

Science.gov (United States)

Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

2015-10-06

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

Live cell CRISPR-imaging in plants reveals dynamic telomere movements

KAUST Repository

Dreissig, Steven

2017-05-16

Elucidating the spatio-temporal organization of the genome inside the nucleus is imperative to understand the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies which reveal genomic information and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm within 30 minutes during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for imaging of multiple genomic loci in live plants cells. CRISPR-imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

Real-time monitoring of caspase cascade activation in living cells.

Science.gov (United States)

Zhu, Lei; Huang, Xinglu; Choi, Ki Young; Ma, Ying; Zhang, Fan; Liu, Gang; Lee, Seulki; Chen, Xiaoyuan

2012-10-10

We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery. Published by Elsevier B.V.

Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes.

Directory of Open Access Journals (Sweden)

Marzia Massignani

Full Text Available BACKGROUND: Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. PRINCIPAL FINDINGS: We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. SIGNIFICANCE: We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation.

Raman microscopy of individual living human embryonic stem cells

DEFF Research Database (Denmark)

Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.

2010-01-01

We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...

Visualization and targeted disruption of protein interactions in living cells

Science.gov (United States)

Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

2013-01-01

Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. PMID:24154492

Functional Nanoporous Polymers from Block Copolymer Precursors

DEFF Research Database (Denmark)

Guo, Fengxiao

Abstract Self-assembly of block copolymers provides well-defined morphologies with characteristic length scales in the nanometer range. Nanoporous polymers prepared by selective removal of one block from self-assembled block copolymers offer great technological promise due to their many potential...... functionalities remains a great challenge due to the limitation of available polymer synthesis and the nanoscale confinement of the porous cavities. The main topic of this thesis is to develop methods for fabrication of functional nanoporous polymers from block copolymer precursors. A method has been developed......, where living anionic polymerization and atom transfer radical polymerization (ATRP) are combined to synthesize a polydimethylsiloxane-b-poly(tert-butyl acrylate)-b-polystyrene (PDMS-b-PtBA-b-PS) triblock copolymer precursor. By using either anhydrous hydrogen fluoride or trifluoroacetic acid, PtBA block...

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 → S transition

International Nuclear Information System (INIS)

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay; Sarvepalli, Kavitha; Sadhale, Parag P.; Nath, Utpal

2011-01-01

Highlights: → TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. → TCP4 expression in yeast retards cell division by blocking G1 → S transition. → Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 → S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 → S arrest is discussed.

Live-cell visualization of gasdermin D-driven pyroptotic cell death.

Science.gov (United States)

Rathkey, Joseph K; Benson, Bryan L; Chirieleison, Steven M; Yang, Jie; Xiao, Tsan S; Dubyak, George R; Huang, Alex Y; Abbott, Derek W

2017-09-01

Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

Science.gov (United States)

Harder, Nathalie; Mora-Bermúdez, Felipe; Godinez, William J; Wünsche, Annelie; Eils, Roland; Ellenberg, Jan; Rohr, Karl

2009-11-01

Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

Poly[(3-hexylthiophene-block-(3-semifluoroalkylthiophene] for Polymer Solar Cells

Directory of Open Access Journals (Sweden)

Takeshi Toru

2010-12-01

Full Text Available We report the synthesis of poly[(3-hexylthiophene-block-(3-(4,4,5,5,6,6,7,7,7-nonafluoroheptylthiophene], P(3HT-b-3SFT, carried out by the Grignard Metathesis Method (GRIM. The copolymers composition was determined by 1H and 19F NMR spectroscopies, and gel permeation chromatography (GPC. The thin films of P(3HT‑b‑3SFT were investigated by ultraviolet-visible absorption spectroscopy and atomic force microscopy (AFM. We also fabricated bulk-hetero junction (BHJ solar cells based on blends of P(3HT-b-3SFT and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM. Although the composition ratio of P3SFT in P(3HT-b-3SFT was low, the influence of P3SFT on the morphology and properties of solar cells was significant. The annealing process for the BHJ solar cells induced the formation of large domains and led to poor solar cell performance. The BHJ solar cells, based on PCBM and P(3HT-b-3SFT, prepared by the non-annealing process, had a maximum power conversion efficiency of 0.84% under 100 mW/cm2 (AM 1.5 solar illumination in air.

Organic photovoltaic cell incorporating electron conducting exciton blocking layers

Science.gov (United States)

Forrest, Stephen R.; Lassiter, Brian E.

2014-08-26

The present disclosure relates to photosensitive optoelectronic devices including a compound blocking layer located between an acceptor material and a cathode, the compound blocking layer including: at least one electron conducting material, and at least one wide-gap electron conducting exciton blocking layer. For example, 3,4,9,10 perylenetetracarboxylic bisbenzimidazole (PTCBI) and 1,4,5,8-napthalene-tetracarboxylic-dianhydride (NTCDA) function as electron conducting and exciton blocking layers when interposed between the acceptor layer and cathode. Both materials serve as efficient electron conductors, leading to a fill factor as high as 0.70. By using an NTCDA/PTCBI compound blocking layer structure increased power conversion efficiency is achieved, compared to an analogous device using a conventional blocking layers shown to conduct electrons via damage-induced midgap states.

Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

Directory of Open Access Journals (Sweden)

Federico Baltar

2018-01-01

Full Text Available Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached, and dissolved (i.e., cell-free enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100% of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles.

Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

Science.gov (United States)

Baltar, Federico

2018-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

Distinct apoptotic blocks mediate resistance to panHER inhibitors in HER2+ breast cancer cells.

Science.gov (United States)

Karakas, Bahriye; Ozmay, Yeliz; Basaga, Huveyda; Gul, Ozgur; Kutuk, Ozgur

2018-05-04

Despite the development of novel targeted therapies, de novo or acquired chemoresistance remains a significant factor for treatment failure in breast cancer therapeutics. Neratinib and dacomitinib are irreversible panHER inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2-overexpressing cell lines. Here we showed that increased MCL1 and decreased BIM and PUMA mediated resistance to neratinib in ZR-75-30 and SKBR3 cells while increased BCL-XL and BCL-2 and decreased BIM and PUMA promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted antiapoptotic protein dependence and development of resistance to panHER inhibitors. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR-75-30 cells. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in neratinib-resistant SKBR3 and ZR-75-30 cells, but we did not detect a similar response in neratinib-resistant BT474 cells. Accordingly, suppression of BCL-2/BCL-XL by ABT-737 was required in addition to ERK1/2 inhibition for neratinib- or dacomitinib-induced apoptosis in neratinib-resistant BT474 cells. Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER inhibitor resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER inhibitor resistance in breast cancer cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Vital Autofluorescence: Application to the Study of Plant Living Cells

Directory of Open Access Journals (Sweden)

Victoria V. Roshchina

2012-01-01

approach to study the autofluorescence of plant living cells—from cell diagnostics up to modelling the cell-cell contacts and cell interactions with fluorescent biologically active substances. It bases on the direct observations of secretions released from allelopathic and medicinal species and the cell-donor interactions with cell-acceptors as biosensors (unicellular plant generative and vegetative microspores. Special attention was paid to the interactions with pigmented and fluorescing components of the secretions released by the cells-donors from plant species. Colored components of secretions are considered as histochemical dyes for the analysis of cellular mechanisms at the cell-cell contacts and modelling of cell-cell interactions. The fluorescence of plant biosensors was also recommended for the testing of natural plant excretions as medical drugs.

Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells

International Nuclear Information System (INIS)

Mas, Josep; Berg-Sørensen, Kirstine; Richardson, Andrew C; Reihani, S Nader S; Oddershede, Lene B

2013-01-01

With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes into account the viscoelastic properties of the cytoplasm and relies on a combination of active and passive recordings of the motion of the cytoplasmic object of interest. The calibration procedure allows us to extract absolute values for the viscoelastic moduli of the living cell cytoplasm as well as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles. The viscoelastic moduli that we find are of the same order of magnitude as moduli found in other cell types by alternative methods. (paper)

Performance enhancement of perovskite solar cells with Mg-doped TiO2 compact film as the hole-blocking layer

International Nuclear Information System (INIS)

Wang, Jing; Qin, Minchao; Tao, Hong; Ke, Weijun; Chen, Zhao; Wan, Jiawei; Qin, Pingli; Lei, Hongwei; Fang, Guojia; Xiong, Liangbin; Yu, Huaqing

2015-01-01

In this letter, we report perovskite solar cells with thin dense Mg-doped TiO 2 as hole-blocking layers (HBLs), which outperform cells using TiO 2 HBLs in several ways: higher open-circuit voltage (V oc ) (1.08 V), power conversion efficiency (12.28%), short-circuit current, and fill factor. These properties improvements are attributed to the better properties of Mg-modulated TiO 2 as compared to TiO 2 such as better optical transmission properties, upshifted conduction band minimum (CBM) and downshifted valence band maximum (VBM), better hole-blocking effect, and higher electron life time. The higher-lying CBM due to the modulation with wider band gap MgO and the formation of magnesium oxide and magnesium hydroxides together resulted in an increment of V oc . In addition, the Mg-modulated TiO 2 with lower VBM played a better role in the hole-blocking. The HBL with modulated band position provided better electron transport and hole blocking effects within the device

Intracellular imaging of docosanol in living cells by coherent anti-Stokes Raman scattering microscopy

Science.gov (United States)

You, Sixian; Liu, Yuan; Arp, Zane; Zhao, Youbo; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

2017-07-01

Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.

Fabrication of honeycomb-structured poly(ethylene glycol)-block-poly(lactic acid) porous films and biomedical applications for cell growth

Energy Technology Data Exchange (ETDEWEB)

Yao, Bingjian [Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250199 (China); College of chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014 (China); Zhu, Qingzeng, E-mail: qzzhu@sdu.edu.cn [Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250199 (China); Yao, Linli [Key Laboratory of the Ministry of Education for Experimental Teratology, Department of Histology and Embryology, Shandong University School of Medicine, 250012 Jinan (China); Hao, Jingcheng [Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250199 (China)

2015-03-30

Graphical abstract: - Highlights: • Honeycomb-structured PEG-PLA porous films were fabricated. • The organization of pores depends on molecular weight ratio of PEG-to-PLA block. • The pores in the film were internally decorated with a layer of PEG. • The honeycomb-structured PEG-PLA film was suitable as a substrate for cell growth. - Abstract: A series of poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) copolymers with a hydrophobic PLA block of different molecular weights and a fixed length hydrophilic PEG were synthesized successfully and characterized. These amphiphilic block copolymers were used to fabricate honeycomb-structured porous films using the breath figure (BF) templating technique. The surface topology and composition of the highly ordered pattern film were further characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy. The results indicated that the PEG-to-PLA block molecular weight ratio influenced the BF film surface topology. The film with the best ordered pores was obtained with a PEG-to-PLA ratio of 2.0 × 10{sup 3}:3.0 × 10{sup 4}. The self-organization of the hydrophilic PEG chains within the pores was confirmed by XPS and fluorescence labeled PEG. A model is proposed to elucidate the stabilization process of the amphiphilic PEG-PLA aggregated architecture on the water droplet-based templates. In addition, GFP-U87 cell viability has been investigated by MTS test and the cell morphology on the honeycomb-structured PEG-PLA porous film has been evaluated using phase-contrast microscope. This porous film is shown to be suitable as a matrix for cell growth.

Fabrication of honeycomb-structured poly(ethylene glycol)-block-poly(lactic acid) porous films and biomedical applications for cell growth

International Nuclear Information System (INIS)

Yao, Bingjian; Zhu, Qingzeng; Yao, Linli; Hao, Jingcheng

2015-01-01

Graphical abstract: - Highlights: • Honeycomb-structured PEG-PLA porous films were fabricated. • The organization of pores depends on molecular weight ratio of PEG-to-PLA block. • The pores in the film were internally decorated with a layer of PEG. • The honeycomb-structured PEG-PLA film was suitable as a substrate for cell growth. - Abstract: A series of poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) copolymers with a hydrophobic PLA block of different molecular weights and a fixed length hydrophilic PEG were synthesized successfully and characterized. These amphiphilic block copolymers were used to fabricate honeycomb-structured porous films using the breath figure (BF) templating technique. The surface topology and composition of the highly ordered pattern film were further characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy. The results indicated that the PEG-to-PLA block molecular weight ratio influenced the BF film surface topology. The film with the best ordered pores was obtained with a PEG-to-PLA ratio of 2.0 × 10 3 :3.0 × 10 4 . The self-organization of the hydrophilic PEG chains within the pores was confirmed by XPS and fluorescence labeled PEG. A model is proposed to elucidate the stabilization process of the amphiphilic PEG-PLA aggregated architecture on the water droplet-based templates. In addition, GFP-U87 cell viability has been investigated by MTS test and the cell morphology on the honeycomb-structured PEG-PLA porous film has been evaluated using phase-contrast microscope. This porous film is shown to be suitable as a matrix for cell growth

Modulation of protein properties in living cells using nanobodies.

Science.gov (United States)

Kirchhofer, Axel; Helma, Jonas; Schmidthals, Katrin; Frauer, Carina; Cui, Sheng; Karcher, Annette; Pellis, Mireille; Muyldermans, Serge; Casas-Delucchi, Corella S; Cardoso, M Cristina; Leonhardt, Heinrich; Hopfner, Karl-Peter; Rothbauer, Ulrich

2010-01-01

Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

An integrated on-line irradiation and in situ live cell imaging system

Energy Technology Data Exchange (ETDEWEB)

Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen, E-mail: gen.yang@pku.edu.cn; Wang, Yugang

2015-09-01

Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO{sub 2}, O{sub 2} concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia.

An integrated on-line irradiation and in situ live cell imaging system

International Nuclear Information System (INIS)

Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen; Wang, Yugang

2015-01-01

Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO 2 , O 2 concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia

An integrated on-line irradiation and in situ live cell imaging system

Science.gov (United States)

Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen; Wang, Yugang

2015-09-01

Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO2, O2 concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia.

Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

Directory of Open Access Journals (Sweden)

Coisne Caroline

2013-01-01

Full Text Available Abstract Background The central nervous system (CNS is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood–brain barrier (BBB located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB. Methods and design An in vitro mouse BBB model maintaining physiological barrier characteristics in a flow chamber and combined with high magnification live cell imaging, has been established. This model enables the molecular mechanisms involved in the multi-step extravasation of T cells across the in vitro BBB, to be defined with high-throughput analyses. Subsequently these mechanisms have been verified in vivo using a limited number of experimental animals and a spinal cord window surgical technique. The window enables live observation of the dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions using real time epifluorescence intravital imaging. These in vitro and in vivo live cell imaging methods have shown that the BBB endothelium possesses unique and specialized mechanisms involved in the multi-step T cell migration across this endothelial barrier under physiological flow. The initial T cell interaction with the endothelium is either mediated by T cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4+ T cells crawl over long distances against the direction of

Thermally induced structural evolution and performance of mesoporous block copolymer-directed alumina perovskite solar cells.

KAUST Repository

Tan, Kwan Wee

2014-04-11

Structure control in solution-processed hybrid perovskites is crucial to design and fabricate highly efficient solar cells. Here, we utilize in situ grazing incidence wide-angle X-ray scattering and scanning electron microscopy to investigate the structural evolution and film morphologies of methylammonium lead tri-iodide/chloride (CH3NH3PbI(3-x)Cl(x)) in mesoporous block copolymer derived alumina superstructures during thermal annealing. We show the CH3NH3PbI(3-x)Cl(x) material evolution to be characterized by three distinct structures: a crystalline precursor structure not described previously, a 3D perovskite structure, and a mixture of compounds resulting from degradation. Finally, we demonstrate how understanding the processing parameters provides the foundation needed for optimal perovskite film morphology and coverage, leading to enhanced block copolymer-directed perovskite solar cell performance.

Thermally induced structural evolution and performance of mesoporous block copolymer-directed alumina perovskite solar cells.

KAUST Repository

Tan, Kwan Wee; Moore, David T; Saliba, Michael; Sai, Hiroaki; Estroff, Lara A; Hanrath, Tobias; Snaith, Henry J; Wiesner, Ulrich

2014-01-01

Structure control in solution-processed hybrid perovskites is crucial to design and fabricate highly efficient solar cells. Here, we utilize in situ grazing incidence wide-angle X-ray scattering and scanning electron microscopy to investigate the structural evolution and film morphologies of methylammonium lead tri-iodide/chloride (CH3NH3PbI(3-x)Cl(x)) in mesoporous block copolymer derived alumina superstructures during thermal annealing. We show the CH3NH3PbI(3-x)Cl(x) material evolution to be characterized by three distinct structures: a crystalline precursor structure not described previously, a 3D perovskite structure, and a mixture of compounds resulting from degradation. Finally, we demonstrate how understanding the processing parameters provides the foundation needed for optimal perovskite film morphology and coverage, leading to enhanced block copolymer-directed perovskite solar cell performance.

Thermally Induced Structural Evolution and Performance of Mesoporous Block Copolymer-Directed Alumina Perovskite Solar Cells

Science.gov (United States)

2015-01-01

Structure control in solution-processed hybrid perovskites is crucial to design and fabricate highly efficient solar cells. Here, we utilize in situ grazing incidence wide-angle X-ray scattering and scanning electron microscopy to investigate the structural evolution and film morphologies of methylammonium lead tri-iodide/chloride (CH3NH3PbI3–xClx) in mesoporous block copolymer derived alumina superstructures during thermal annealing. We show the CH3NH3PbI3–xClx material evolution to be characterized by three distinct structures: a crystalline precursor structure not described previously, a 3D perovskite structure, and a mixture of compounds resulting from degradation. Finally, we demonstrate how understanding the processing parameters provides the foundation needed for optimal perovskite film morphology and coverage, leading to enhanced block copolymer-directed perovskite solar cell performance. PMID:24684494

Examining live cell cultures during apoptosis by digital holographic phase imaging and Raman spectroscopy

Science.gov (United States)

Khmaladze, Alexander

2017-11-01

Cellular apoptosis is a unique, organized series of events, leading to programmed cell death. In this work, we present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy measurements of live cell cultures yield information about cell shape and volume, changes to which are indicative of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopic measurements provide complementary information about cells, such as protein, lipid and nucleic acid content, and the spectral signatures associated with structural changes in molecules. Our work indicates that the chemical changes in proteins, which were detected by Raman measurements, preceded morphological changes, which were seen with digital holographic microscopy.

IL-15 induces strong but short-lived tumor-infiltrating CD8 T cell responses through the regulation of Tim-3 in breast cancer

Energy Technology Data Exchange (ETDEWEB)

Heon, Elise K. [University of Maryland Medical Center, Baltimore, MD 21201 (United States); Wulan, Hasi [Department of Plastic and Reconstructive Surgery, PLA General Hospital, Beijing, 100853 (China); Macdonald, Loch P.; Malek, Adel O.; Braunstein, Glenn H.; Eaves, Connie G.; Schattner, Mark D. [Brown University, Providence, RI 02912 (United States); Allen, Peter M.; Alexander, Michael O.; Hawkins, Cynthia A.; McGovern, Dermot W.; Freeman, Richard L. [University of Wisconsin, Madison, WI 53706 (United States); Amir, Eitan P.; Huse, Jason D. [University of Illinois, Chicago, IL 60607 (United States); Zaltzman, Jeffrey S.; Kauff, Noah P.; Meyers, Paul G. [University of Texas, Austin, TX 78712 (United States); Gleason, Michelle H., E-mail: GleasonM@cblabs.org [University of Texas, Austin, TX 78712 (United States); Overholtzer, Michael G., E-mail: OverholtzerM@cblabs.org [University of Texas, Austin, TX 78712 (United States); Wiseman, Sam S. [Ohio State University, Columbus, OH 43210 (United States); and others

2015-08-14

and IL-2 had different kinetics in inducing TI CD8 T cell responses. • IL-15 induced stronger but shorter-lived TI CD8 T cell responses than IL-2. • IL-15, but not IL-2, caused upregulation of Tim-3 on TI CD8 T cells. • Blocking Tim-3 resulted in increased IL-15-induced proliferation and IFN-γ production in TI CD8 T cells.

IL-4 production by group 2 innate lymphoid cells promotes food allergy by blocking regulatory T-cell function.

Science.gov (United States)

Noval Rivas, Magali; Burton, Oliver T; Oettgen, Hans C; Chatila, Talal

2016-09-01

Food allergy is a major health issue, but its pathogenesis remains obscure. Group 2 innate lymphoid cells (ILC2s) promote allergic inflammation. However their role in food allergy is largely unknown. We sought to investigate the role of ILC2s in food allergy. Food allergy-prone mice with a gain-of-function mutation in the IL-4 receptor α chain (Il4raF709) were orally sensitized with food allergens, and the ILC2 compartment was analyzed. The requirement for ILC2s in food allergy was investigated by using Il4raF709, IL-33 receptor-deficient (Il1rl1(-/-)), IL-13-deficient (Il13(-/-)), and IL-4-deficient (Il4(-/-)) mice and by adoptive transfer of in vitro-expanded ILC2s. Direct effects of ILC2s on regulatory T (Treg) cells and mast cells were analyzed in coculture experiments. Treg cell control of ILC2s was assessed in vitro and in vivo. Il4raF709 mice with food allergy exhibit increased numbers of ILC2s. IL-4 secretion by ILC2s contributes to the allergic response by reducing allergen-specific Treg cell and activating mast cell counts. IL-33 receptor deficiency in Il4raF709 Il1rl1(-/-) mice protects against allergen sensitization and anaphylaxis while reducing ILC2 induction. Adoptive transfer of wild-type and Il13(-/-) but not Il4(-/-) ILC2s restored sensitization in Il4raF709 Il1rl1(-/-) mice. Treg cells suppress ILC2s in vitro and in vivo. IL-4 production by IL-33-stimulated ILC2s blocks the generation of allergen-specific Treg cells and favors food allergy. Strategies to block ILC2 activation or the IL-33/IL-33 receptor pathway can lead to innovative therapies in the treatment of food allergy. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

Science.gov (United States)

Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

2012-05-01

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

Raman tweezers spectroscopy of live, single red and white blood cells.

Directory of Open Access Journals (Sweden)

Aseefhali Bankapur

Full Text Available An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC and white blood cells (WBC under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW. Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.

Energy, control and DNA structure in the living cell

DEFF Research Database (Denmark)

Wijker, J.E.; Jensen, Peter Ruhdal; Gomes, A. Vaz

1995-01-01

Maintenance (let alone growth) of the highly ordered living cell is only possible through the continuous input of free energy. Coupling of energetically downhill processes (such as catabolic reactions) to uphill processes is essential to provide this free energy and is catalyzed by enzymes either...

Detecting protein-protein interactions in living cells

DEFF Research Database (Denmark)

Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

2009-01-01

to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C......-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat......The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared...

Detection and analysis of human serum albumin nanoparticles within phagocytic cells at the resolution of individual live cell or single 3D multicellular spheroid

Energy Technology Data Exchange (ETDEWEB)

Afrimzon, Elena; Zurgil, Naomi; Sobolev, Maria; Shafran, Yana [Bar-Ilan University, The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome (Israel); Langer, Klaus; Zlatev, Iavor [Westfälischen Wilhelms-Universität Münster, Institut für Pharmazeutische Technologie und Biopharmazie (Germany); Wronski, Robert; Windisch, Manfred [QPS Austria GmbH (Austria); Briesen, Hagen von [Fraunhofer Institute for Biomedical Engineering IBMT, Department of Cell Biology & Applied Virology (Germany); Schmidt, Reinhold [Medical University of Graz, Department of Neurology (Austria); Pietrzik, Claus [University Medical Center of the Johannes Gutenberg University of Mainz, Institute of Pathobiochemistry (Germany); Deutsch, Mordechai, E-mail: motti.jsc@gmail.com [Bar-Ilan University, The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome (Israel)

2015-12-15

Since nanoparticles (NPs) have shown great potential in various biomedical applications, live cell response to NPs should be thoroughly explored prior to their in vivo use. In the current study, live cell array (LCA) methodology and unique cell-based assays were used to study the interaction of magnetite (HSA-Mag NP) loaded human serum albumin NPs with phagocytic cells. The LCA enabled cell culturing during HSA-Mag NP accumulation and monolayer or spheroid formation, concomitantly with on-line monitoring of NP internalization. These platforms were also utilized for imaging intercellular links between living cells preloaded with HSA-Mag NP in 2D and 3D resolution. HSA-Mag NP uptake by cells was quantified by imaging, and analyzed using time-resolved measurements. Image analysis of the individual cells in cell populations showed accumulation of HSA-Mag NP by promonocytes and glial cells in a dose- and time-dependent manner. High variability of NP accumulation in individual cells within cell populations, as well as cell subgroups, was evident in both cell types. Following 24 h interaction, uptake of HSA-Mag NP was about 10 times more efficient in glial cells than in activated promonocytes. The presented assays may facilitate detection and analysis of the amount of NPs within individual cells, as well as the rate of NP accumulation and processing in different subsets of living cells. Such data are crucial for estimating predicted drug dosage delivered by NPs, as well as to study possible mechanisms for NP interference with live cells.

Optical detection and virotherapy of live metastatic tumor cells in body fluids with vaccinia strains.

Directory of Open Access Journals (Sweden)

Huiqiang Wang

Full Text Available Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV. In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.

Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

Science.gov (United States)

Yerramilli, V Siddartha; Kim, Kyung Hyuk

2018-03-16

RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

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Jun Wang

2012-04-01

Full Text Available Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA, the electric cell-substrate impedance sensing (ECIS technique, and the light addressable potentiometric sensor (LAPS. The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology.

Live cell imaging reveals marked variability in myoblast proliferation and fate

Science.gov (United States)

2013-01-01

Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

Methotrexate-Loaded Four-Arm Star Amphiphilic Block Copolymer Elicits CD8+ T Cell Response against a Highly Aggressive and Metastatic Experimental Lymphoma.

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Hira, Sumit Kumar; Ramesh, Kalyan; Gupta, Uttam; Mitra, Kheyanath; Misra, Nira; Ray, Biswajit; Manna, Partha Pratim

2015-09-16

We have synthesized a well-defined four-arm star amphiphilic block copolymer [poly(DLLA)-b-poly(NVP)]4 [star-(PDLLA-b-PNVP)4] that consists of D,L-lactide (DLLA) and N-vinylpyrrolidone (NVP) via the combination of ring-opening polymerization (ROP) and xanthate-mediated reversible addition-fragmentation chain transfer (RAFT) polymerization. Synthesis of the polymer was verified by 1H NMR spectroscopy and gel permeation chromatography (GPC). The amphiphilic four-arm star block copolymer forms spherical micelles in water as demonstrated by transmission electron microscopy (TEM) and 1H NMR spectroscopy. Pyrene acts as a probe to ascertain the critical micellar concentration (cmc) by using fluorescence spectroscopy. Methotrexate (MTX)-loaded polymeric micelles of star-(PDLLA15-b-PNVP10)4 amphiphilic block copolymer were prepared and characterized by fluorescence and TEM studies. Star-(PDLLA15-b-PNVP10)4 copolymer was found to be significantly effective with respect to inhibition of proliferation and lysis of human and murine lymphoma cells. The amphiphilic block copolymer causes cell death in parental and MTX-resistant Dalton lymphoma (DL) and Raji cells. The formulation does not cause hemolysis in red blood cells and is tolerant to lymphocytes compared to free MTX. Therapy with MTX-loaded star-(PDLLA15-b-PNVP10)4 amphiphilic block copolymer micelles prolongs the life span of animals with neoplasia by reducing the tumor load, preventing metastasis and augmenting CD8+ T cell-mediated adaptive immune responses.

Assessment of FUN-1 vital dye staining: Yeast with a block in the vacuolar sorting pathway have impaired ability to form CIVS when stained with FUN-1 fluorescent dye.

Science.gov (United States)

Essary, Brandin D; Marshall, Pamela A

2009-08-01

FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide] is a fluorescent dye used in studies of yeast and other fungi to monitor cell viability in the research lab and to assay for active fungal infection in the clinical setting. When the plasma membrane is intact, fungal cells internalize FUN-1 and the dye is seen as diffuse green cytosolic fluorescence. FUN-1 is then transported to the vacuole in metabolically active wild type cells and subsequently is compacted into fluorescent red cylindrical intravacuolar structures (CIVS) by an unknown transport pathway. This dye is used to determine yeast viability, as only live cells form CIVS. However, in live Saccharomyces cerevisiae with impaired protein sorting to the yeast vacuole, we report decreased to no CIVS formation, depending on the cellular location of the block in the sorting pathway. Cells with a block in vesicle-mediated transport from the Golgi to prevacuolar compartment (PVC) or with a block in recycling from the PVC to the Golgi demonstrate a substantial impairment in CIVS formation. Instead, the FUN-1 dye is seen either in small punctate structures under fluorescence or as diffuse red cytosol under white light. Thus, researchers using FUN-1 should be cognizant of the limitations of this procedure in determining cell viability as there are viable yeast mutants with impaired CIVS formation.

On strain and stress in living cells

Science.gov (United States)

Cox, Brian N.; Smith, David W.

2014-11-01

Recent theoretical simulations of amelogenesis and network formation and new, simple analyses of the basic multicellular unit (BMU) allow estimation of the order of magnitude of the strain energy density in populations of living cells in their natural environment. A similar simple calculation translates recent measurements of the force-displacement relation for contacting cells (cell-cell adhesion energy) into equivalent volume energy densities, which are formed by averaging the changes in contact energy caused by a cell's migration over the cell's volume. The rates of change of these mechanical energy densities (energy density rates) are then compared to the order of magnitude of the metabolic activity of a cell, expressed as a rate of production of metabolic energy per unit volume. The mechanical energy density rates are 4-5 orders of magnitude smaller than the metabolic energy density rate in amelogenesis or bone remodeling in the BMU, which involve modest cell migration velocities, and 2-3 orders of magnitude smaller for innervation of the gut or angiogenesis, where migration rates are among the highest for all cell types. For representative cell-cell adhesion gradients, the mechanical energy density rate is 6 orders of magnitude smaller than the metabolic energy density rate. The results call into question the validity of using simple constitutive laws to represent living cells. They also imply that cells need not migrate as inanimate objects of gradients in an energy field, but are better regarded as self-powered automata that may elect to be guided by such gradients or move otherwise. Thus Ġel=d/dt 1/2 >[(C11+C12)ɛ02+2μγ02]=(C11+C12)ɛ0ɛ˙0+2μγ0γ˙0 or Ġel=ηEɛ0ɛ˙0+η‧Eγ0γ˙0 with 1.4≤η≤3.4 and 0.7≤η‧≤0.8 for Poisson's ratio in the range 0.2≤ν≤0.4 and η=1.95 and η‧=0.75 for ν=0.3. The spatial distribution of shear strains arising within an individual cell as cells slide past one another during amelogenesis is not known

TimeLapseAnalyzer: Multi-target analysis for live-cell imaging and time-lapse microscopy

DEFF Research Database (Denmark)

Huth, Johannes; Buchholz, Malte; Kraus, Johann M.

2011-01-01

The direct observation of cells over time using time-lapse microscopy can provide deep insights into many important biological processes. Reliable analyses of motility, proliferation, invasive potential or mortality of cells are essential to many studies involving live cell imaging and can aid in...... counting and tube formation analysis in high throughput screening of live-cell experiments. TimeLapseAnalyzer is freely available (MATLAB, Open Source) at http://www.informatik.uniulm. de/ni/mitarbeiter/HKestler/tla......., we developed TimeLapseAnalyzer. Apart from general purpose image enhancements and segmentation procedures, this extensible, self-contained, modular cross-platform package provides dedicated modalities for fast and reliable analysis of multi-target cell tracking, scratch wound healing analysis, cell...

The radiation effects on the living cell

International Nuclear Information System (INIS)

Sage, E.; Dutrillaux, B.; Soussi, Th.; Boiteux, S.; Lopez, B.; Feunteun, J.

1999-06-01

This publication is a presentation of particular points discussed during the colloquium of the 15-18 june 1999, for which scientific researches are performed at the CEA/CNRS. They deal with the radiobiology, for the radiation effects on living matter; with the DNA, for the knowledge and repair mechanisms on cells submitted to ionizing radiations; with the exposition to UV in correlation with neoplasms; with the P53 gene which is a tumour suppressor. (A.L.B.)

Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

Science.gov (United States)

Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

2015-02-11

Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

Living with a diagnosis of non-small cell lung cancer: patients' lived experiences.

LENUS (Irish Health Repository)

McCarthy, Ita

2012-01-31

The aim of this study was to explore patients\\' experience of living with non-small cell lung cancer (NSCLC). Patients diagnosed with NSCLC know that their treatment is not with curative intent and can expect distressing symptoms. In this phenomenological study, six adults with a diagnosis of NSCLC were interviewed. Data was analysed guided by van Manen\\'s six-step process. Four main themes were interpreted: \\'Maintaining my life\\'; \\'The enemy within\\'; \\'Staying on the train\\

Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

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Viacheslav O. Nikolaev

2013-04-01

Full Text Available Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains.

Secondary Metabolite Localization by Autofluorescence in Living Plant Cells

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Pascale Talamond

2015-03-01

Full Text Available Autofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla. This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment.

In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells.

Science.gov (United States)

Chen, H; Ouyang, W; Jones, M; Haque, T; Lawuyi, B; Prakash, S

2005-08-01

Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

Suitability of the Cellient (TM) cell block method for diagnosing soft tissue and bone tumors

NARCIS (Netherlands)

Song, W.; van Hemel, B. M.; Suurmeijer, A. J. H.

BACKGROUNDThe diagnosis of tumors of soft tissue and bone (STB) heavily relies on histological biopsies, whereas cytology is not widely used. Cellient(TM) cell blocks often contain small tissue fragments. In addition to Hematoxylin and Eosin (H&E) interpretation of histological features,

The live cell irradiation and observation setup at SNAKE

Energy Technology Data Exchange (ETDEWEB)

Hable, V. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany)], E-mail: volker.hable@unibw.de; Greubel, C.; Bergmaier, A.; Reichart, P. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany); Hauptner, A.; Kruecken, R. [Physik Department E12, TU-Muenchen, 85748 Garching (Germany); Strickfaden, H.; Dietzel, S.; Cremer, T. [Department Biologie II, LMU-Muenchen, 82152 Martinsried (Germany); Drexler, G.A.; Friedl, A.A. [Strahlenbiologisches Institut, LMU-Muenchen, 80336 Muenchen (Germany); Dollinger, G. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany)

2009-06-15

We describe a new setup at the ion microprobe SNAKE (Superconducting Nanoscope for Applied nuclear (Kern-) physics Experiments) at the Munich 14 MV Tandem accelerator that facilitates both living cell irradiation with sub micrometer resolution and online optical imaging of the cells before and after irradiation by state of the art phase contrast and fluorescence microscopy. The cells are kept at standard cell growth conditions at 37 {sup o}C in cell culture medium. After irradiation it is possible to switch from single ion irradiation conditions to cell observation within 0.5 s. First experiments were performed targeting substructures of a cell nucleus that were tagged by TexasRed labeled nucleotides incorporated in the cellular DNA by 55 MeV single carbon ion irradiation. In addition we show first online sequences of short time kinetics of Mdc1 protein accumulation in the vicinity of double strand breaks after carbon ion irradiation.

The 3' untranslated region of the cyclin B mRNA is not sufficient to enhance the synthesis of cyclin B during a mitotic block in human cells.

Directory of Open Access Journals (Sweden)

Dominik Schnerch

Full Text Available Antimitotic agents are frequently used to treat solid tumors and hematologic malignancies. However, one major limitation of antimitotic approaches is mitotic slippage, which is driven by slow degradation of cyclin B during a mitotic block. The extent to which cyclin B levels decline is proposed to be governed by an equilibrium between cyclin B synthesis and degradation. It was recently shown that the 3' untranslated region (UTR of the murine cyclin B mRNA contributes to the synthesis of cyclin B during mitosis in murine cells. Using a novel live-cell imaging-based technique allowing us to study synthesis and degradation of cyclin B simultaneously at the single cell level, we tested here the role of the human cyclin B 3'UTR in regulating cyclin B synthesis during mitosis in human cells. We observed that the cyclin B 3'UTR was not sufficient to enhance cyclin B synthesis in human U2Os, HeLa or hTERT RPE-1 cells. A better understanding of how the equilibrium of cyclin B is regulated in mitosis may contribute to the development of improved therapeutic approaches to prevent mitotic slippage in cancer cells treated with antimitotic agents.

Minocycline blocks glial cell activation and ventilatory acclimatization to hypoxia.

Science.gov (United States)

Stokes, Jennifer A; Arbogast, Tara E; Moya, Esteban A; Fu, Zhenxing; Powell, Frank L

2017-04-01

Ventilatory acclimatization to hypoxia (VAH) is the time-dependent increase in ventilation, which persists upon return to normoxia and involves plasticity in both central nervous system respiratory centers and peripheral chemoreceptors. We investigated the role of glial cells in VAH in male Sprague-Dawley rats using minocycline, an antibiotic that inhibits microglia activation and has anti-inflammatory properties, and barometric pressure plethysmography to measure ventilation. Rats received either minocycline (45mg/kg ip daily) or saline beginning 1 day before and during 7 days of chronic hypoxia (CH, Pi O 2  = 70 Torr). Minocycline had no effect on normoxic control rats or the hypercapnic ventilatory response in CH rats, but minocycline significantly ( P minocycline administration during only the last 3 days of CH did not reverse VAH. Microglia and astrocyte activation in the nucleus tractus solitarius was quantified from 30 min to 7 days of CH. Microglia showed an active morphology (shorter and fewer branches) after 1 h of hypoxia and returned to the control state (longer filaments and extensive branching) after 4 h of CH. Astrocytes increased glial fibrillary acidic protein antibody immunofluorescent intensity, indicating activation, at both 4 and 24 h of CH. Minocycline had no effect on glia in normoxia but significantly decreased microglia activation at 1 h of CH and astrocyte activation at 24 h of CH. These results support a role for glial cells, providing an early signal for the induction but not maintenance of neural plasticity underlying ventilatory acclimatization to hypoxia. NEW & NOTEWORTHY The signals for neural plasticity in medullary respiratory centers underlying ventilatory acclimatization to chronic hypoxia are unknown. We show that chronic hypoxia activates microglia and subsequently astrocytes. Minocycline, an antibiotic that blocks microglial activation and has anti-inflammatory properties, also blocks astrocyte activation in respiratory

Blocking of PDL-1 interaction enhances primary and secondary CD8 T cell response to herpes simplex virus-1 infection.

Directory of Open Access Journals (Sweden)

Rudragouda Channappanavar

Full Text Available The blocking of programmed death ligand-1 (PDL-1 has been shown to enhance virus-specific CD8 T cell function during chronic viral infections. Though, how PDL-1 blocking at the time of priming affects the quality of CD8 T cell response to acute infections is not well understood and remains controversial. This report demonstrates that the magnitude of the primary and secondary CD8 T cell responses to herpes simplex virus-1 (HSV-1 infection is subject to control by PDL-1. Our results showed that after footpad HSV-1 infection, PD-1 expression increases on immunodominant SSIEFARL peptide specific CD8 T cells. Additionally, post-infection, the level of PDL-1 expression also increases on CD11c+ dendritic cells. Intraperitoneal administration of anti-PDL-1 monoclonal antibody given one day prior to and three days after cutaneous HSV-1 infection, resulted in a marked increase in effector and memory CD8 T cell response to SSIEFARL peptide. This was shown by measuring the quantity and quality of SSIEFARL-specific CD8 T cells by making use of ex-vivo assays that determine antigen specific CD8 T cell function, such as intracellular cytokine assay, degranulation assay to measure cytotoxicity and viral clearance. Our results are discussed in terms of the beneficial effects of blocking PDL-1 interactions, while giving prophylactic vaccines, to generate a more effective CD8 T cell response to viral infection.

PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse.

Science.gov (United States)

Huang, Yu; Chen, Zhiying; Jang, Joon Hee; Baig, Mirza S; Bertolet, Grant; Schroeder, Casey; Huang, Shengjian; Hu, Qian; Zhao, Yong; Lewis, Dorothy E; Qin, Lidong; Zhu, Michael Xi; Liu, Dongfang

2018-04-18

The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

[Construction of porous hydroxyapatite (HA) block loaded with cultured chondrocytes].

Science.gov (United States)

Yan, M; Dang, G

1999-07-01

To construct a kind of bone healing enhancing implant with cultured chondrocytes bound to hydroxyapatite (HA). Chondrocytes were obtained from the costicartilage of rat and were cultured on the porous HA blocks, 3 mm x 3 mm x 4 mm size, for three and seven days. Scanning electron micrograph was taken to show whether the cells grew outside and inside the pore of HA block. The cells cultured on tiny glass sheet for 2 days were used to prove where the cells come from by in situ hybridization technique with alpha1 (II) cDNA probe. Scanning electron micrographs showed that the pores of the HA surface and inside of the blocks are filled with cultured cells, especially the longer cultured block. The cells were chondrocytes confirmed by in situ hybridization. The porous HA can be used as cell cultured substrate and chondrocyte can adhere and proliferate inside the porous HA block.

Castration-Resistant Lgr5+ Cells Are Long-Lived Stem Cells Required for Prostatic Regeneration

Directory of Open Access Journals (Sweden)

Bu-er Wang

2015-05-01

Full Text Available The adult prostate possesses a significant regenerative capacity that is of great interest for understanding adult stem cell biology. We demonstrate that leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5 is expressed in a rare population of prostate epithelial progenitor cells, and a castration-resistant Lgr5+ population exists in regressed prostate tissue. Genetic lineage tracing revealed that Lgr5+ cells and their progeny are primarily luminal. Lgr5+ castration-resistant cells are long lived and upon regeneration, both luminal Lgr5+ cells and basal Lgr5+ cells expand. Moreover, single Lgr5+ cells can generate multilineage prostatic structures in renal transplantation assays. Additionally, Lgr5+ cell depletion revealed that the regenerative potential of the castrated adult prostate depends on Lgr5+ cells. Together, these data reveal insights into the cellular hierarchy of castration-resistant Lgr5+ cells, indicate a requirement for Lgr5+ cells during prostatic regeneration, and identify an Lgr5+ adult stem cell population in the prostate.

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

Science.gov (United States)

Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

2013-08-01

Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

A simple optical fiber device for quantitative fluorescence microscopy of single living cells

NARCIS (Netherlands)

van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

1993-01-01

simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

Science.gov (United States)

MacLean, Lorna; Price, Helen; O'Toole, Peter

2016-01-01

Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

Science.gov (United States)

Balbach, Sebastian T; Boiani, Michele

2015-01-01

Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

Candidate Microbicides Block HIV-1 Infection of Human Immature Langerhans Cells within Epithelial Tissue Explants

Science.gov (United States)

Kawamura, Tatsuyoshi; Cohen, Sandra S.; Borris, Debra L.; Aquilino, Elisabeth A.; Glushakova, Svetlana; Margolis, Leonid B.; Orenstein, Jan M.; Offord, Robin E.; Neurath, A. Robert; Blauvelt, Andrew

2000-01-01

Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1Ba-L infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08–4.77%). HIV-1–infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1–1 μg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1Ba-L (an R5 HIV-1 strain) more efficiently infected LC–T cell cocultures when compared with HIV-1IIIB (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1. PMID:11085750

Complex Macromolecular Architectures by Living Cationic Polymerization

KAUST Repository

Alghamdi, Reem D.

2015-05-01

Poly (vinyl ether)-based graft polymers have been synthesized by the combination of living cationic polymerization of vinyl ethers with other living or controlled/ living polymerization techniques (anionic and ATRP). The process involves the synthesis of well-defined homopolymers (PnBVE) and co/terpolymers [PnBVE-b-PCEVE-b-PSiDEGVE (ABC type) and PSiDEGVE-b-PnBVE-b-PSiDEGVE (CAC type)] by sequential living cationic polymerization of n-butyl vinyl ether (nBVE), 2-chloroethyl vinyl ether (CEVE) and tert-butyldimethylsilyl ethylene glycol vinyl ether (SiDEGVE), using mono-functional {[n-butoxyethyl acetate (nBEA)], [1-(2-chloroethoxy) ethyl acetate (CEEA)], [1-(2-(2-(t-butyldimethylsilyloxy)ethoxy) ethoxy) ethyl acetate (SiDEGEA)]} or di-functional [1,4-cyclohexanedimethanol di(1-ethyl acetate) (cHMDEA), (VEMOA)] initiators. The living cationic polymerizations of those monomers were conducted in hexane at -20 0C using Et3Al2Cl3 (catalyst) in the presence of 1 M AcOEt base.[1] The PCEVE segments of the synthesized block terpolymers were then used to react with living macroanions (PS-DPE-Li; poly styrene diphenyl ethylene lithium) to afford graft polymers. The quantitative desilylation of PSiDEGVE segments by n-Bu4N+F- in THF at 0 °C led to graft co- and terpolymers in which the polyalcohol is the outer block. These co-/terpolymers were subsequently subjected to “grafting-from” reactions by atom transfer radical polymerization (ATRP) of styrene to afford more complex macromolecular architectures. The base assisted living cationic polymerization of vinyl ethers were also used to synthesize well-defined α-hydroxyl polyvinylether (PnBVE-OH). The resulting polymers were then modified into an ATRP macro-initiator for the synthesis of well-defined block copolymers (PnBVE-b-PS). Bifunctional PnBVE with terminal malonate groups was also synthesized and used as a precursor for more complex architectures such as H-shaped block copolymer by “grafting-from” or �

A new fluorescent pH probe for imaging lysosomes in living cells.

Science.gov (United States)

Lv, Hong-Shui; Huang, Shu-Ya; Xu, Yu; Dai, Xi; Miao, Jun-Ying; Zhao, Bao-Xiang

2014-01-15

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5-4.1 with the pKa value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no 'alkalizing effect' on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Photoinducible bioorthogonal chemistry: a spatiotemporally controllable tool to visualize and perturb proteins in live cells.

Science.gov (United States)

Lim, Reyna K V; Lin, Qing

2011-09-20

Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pretagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from. In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole-alkene cycloaddition reaction ("photoclick chemistry") and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M(-1) s(-1)), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short "bridge" between two flanking phenyl rings. This photoclick chemistry has been used

Block copolymer based composition and morphology control in nanostructured hybrid materials for energy conversion and storage: solar cells, batteries, and fuel cells

KAUST Repository

Orilall, M. Christopher; Wiesner, Ulrich

2011-01-01

to materials design and current limitations, the review will highlight some of the most recent examples of block copolymer structure-directed nanomaterials for photovoltaics, batteries and fuel cells. In each case insights are provided into the various

Fully synthetic phage-like system for screening mixtures of small molecules in live cells.

Science.gov (United States)

Byk, Gerardo; Partouche, Shirly; Weiss, Aryeh; Margel, Shlomo; Khandadash, Raz

2010-05-10

A synthetic "phage-like" system was designed for screening mixtures of small molecules in live cells. The core of the system consists of 2 mum diameter cross-linked monodispersed microspheres bearing a panel of fluorescent tags and peptides or small molecules either directly synthesized or covalently conjugated to the microspheres. The microsphere mixtures were screened for affinity to cell line PC-3 (prostate cancer model) by incubation with live cells, and as was with phage-display peptide methods, unbound microspheres were removed by repeated washings followed by total lysis of cells and analysis of the bound microspheres by flow-cytometry. Similar to phage-display peptide screening, this method can be applied even in the absence of prior information about the cellular targets of the candidate ligands, which makes the system especially interesting for selection of molecules with high affinity for desired cells, tissues, or tumors. The advantage of the proposed system is the possibility of screening synthetic non-natural peptides or small molecules that cannot be expressed and screened using phage display libraries. A library composed of small molecules synthesized by the Ugi reaction was screened, and a small molecule, Rak-2, which strongly binds to PC-3 cells was found. Rak-2 was then individually synthesized and validated in a complementary whole cell-based binding assay, as well as by live cell microscopy. This new system demonstrates that a mixture of molecules bound to subcellular sized microspheres can be screened on plated cells. Together with other methods using subcellular sized particles for cellular multiplexing, this method represents an important milestone toward high throughput screening of mixtures of small molecules in live cells and in vivo with potential applications in the fields of drug delivery and diagnostic imaging.

Imaging proteolytic activity in live cells and animal models.

Directory of Open Access Journals (Sweden)

Stefanie Galbán

Full Text Available In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors; their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

Science.gov (United States)

Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

2017-10-01

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

Tracking neuronal marker expression inside living differentiating cells using molecular beacons

DEFF Research Database (Denmark)

Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole

2013-01-01

and tyrosine hydroxylase mRNAs were expressed 2 and 3 days post induction of differentiation, respectively. Oct 4 was not detected with MB in these cells and signal was not increased over time suggesting that MB are generally stable inside the cells. The gene expression changes measured using MBs were...... confirmed using qRT-PCR. These results suggest that MBs are simple to use sensors inside living cell, and particularly useful for studying dynamic gene expression in heterogeneous cell populations....

Use of luciferase probes to measure ATP in living cells and animals.

Science.gov (United States)

Morciano, Giampaolo; Sarti, Alba Clara; Marchi, Saverio; Missiroli, Sonia; Falzoni, Simonetta; Raffaghello, Lizzia; Pistoia, Vito; Giorgi, Carlotta; Di Virgilio, Francesco; Pinton, Paolo

2017-08-01

ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

Energy Technology Data Exchange (ETDEWEB)

Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

2014-04-20

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

Living biointerfaces based on non-pathogenic bacteria to direct cell differentiation

Science.gov (United States)

Rodrigo-Navarro, Aleixandre; Rico, Patricia; Saadeddin, Anas; Garcia, Andres J.; Salmeron-Sanchez, Manuel

2014-07-01

Genetically modified Lactococcus lactis, non-pathogenic bacteria expressing the FNIII7-10 fibronectin fragment as a protein membrane have been used to create a living biointerface between synthetic materials and mammalian cells. This FNIII7-10 fragment comprises the RGD and PHSRN sequences of fibronectin to bind α5β1 integrins and triggers signalling for cell adhesion, spreading and differentiation. We used L. lactis strain to colonize material surfaces and produce stable biofilms presenting the FNIII7-10 fragment readily available to cells. Biofilm density is easily tunable and remains stable for several days. Murine C2C12 myoblasts seeded over mature biofilms undergo bipolar alignment and form differentiated myotubes, a process triggered by the FNIII7-10 fragment. This biointerface based on living bacteria can be further modified to express any desired biochemical signal, establishing a new paradigm in biomaterial surface functionalisation for biomedical applications.

Open-dish incubator for live cell imaging with an inverted microscope.

Science.gov (United States)

Heidemann, Steven R; Lamoureux, Phillip; Ngo, Kha; Reynolds, Matthew; Buxbaum, Robert E

2003-10-01

Here we describe the design and fabrication of an inexpensive cell culture incubator for the stage of an inverted light microscope for use in live cell imaging. This device maintains the temperature of the cell culture at 37 degrees C with great stability and, after reaching equilibrium, provides focal stability of an image for 20-25 min with oil-immersion lenses. We describe two versions of the incubator: one for use with standard 60-mm plastic culture dishes, and the other version for imaging of cells on glass coverslips. Either can be made for less than $400. Most components are widely available commercially, and it requires only simple wiring and 3 h to assemble. Although the device is generally useful for live cell imaging on an inverted microscope, it is particularly suitable for work in which instruments are introduced into the culture, such as electrophysiology or micromanipulation. The design is based on the principle that control performance is limited by the lag time between detection and response. The key element of the design is a heated, temperature-controlled aluminum ring serving as a mini-incubator surrounding the culture vessel. For this reason, we call our design a "ringcubator."

Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

Science.gov (United States)

Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

2009-08-13

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

Layer-by-Layer Formation of Block-Copolymer-Derived TiO2 for Solid-State Dye-Sensitized Solar Cells

KAUST Repository

Guldin, Stefan

2011-12-15

Morphology control on the 10 nm length scale in mesoporous TiO 2 films is crucial for the manufacture of high-performance dye-sensitized solar cells. While the combination of block-copolymer self-assembly with sol-gel chemistry yields good results for very thin films, the shrinkage during the film manufacture typically prevents the build-up of sufficiently thick layers to enable optimum solar cell operation. Here, a study on the temporal evolution of block-copolymer-directed mesoporous TiO 2 films during annealing and calcination is presented. The in-situ investigation of the shrinkage process enables the establishment of a simple and fast protocol for the fabrication of thicker films. When used as photoanodes in solid-state dye-sensitized solar cells, the mesoporous networks exhibit significantly enhanced transport and collection rates compared to the state-of-the-art nanoparticle-based devices. As a consequence of the increased film thickness, power conversion efficiencies above 4% are reached. Fabrication of sufficiently thick mesoporous TiO 2 photoelectrodes with morphology control on the 10 nm length scale is essential for solid-state dye-sensitized solar cells (ss-DSC). This study of the temporal evolution of block-copolymer-directed mesoporous TiO 2 films during annealing and calcination enables the build-up of sufficiently thick films for high-performance ssDSC devices. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate.

Science.gov (United States)

MacIntyre, Hugh L; Cullen, John J

2016-08-01

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5-chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat-killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per-cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live-dead classification was essentially error free. But overlap between the frequency distributions of living and heat-killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat-killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8-10 of 24 species (i.e., 33%-42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone. © 2016 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.

Mitochondria Targeted Nanoscale Zeolitic Imidazole Framework-90 for ATP Imaging in Live Cells.

Science.gov (United States)

Deng, Jingjing; Wang, Kai; Wang, Ming; Yu, Ping; Mao, Lanqun

2017-04-26

Zeolitic imidazole frameworks (ZIFs) are an emerging class of functional porous materials with promising biomedical applications such as molecular sensing and intracellular drug delivery. We report herein the first example of using nanoscale ZIFs (i.e., ZIF-90), self-assembled from Zn 2+ and imidazole-2-carboxyaldehyde, to target subcellular mitochondria and image dynamics of mitochondrial ATP in live cells. Encapsulation of fluorescent Rhodamine B (RhB) into ZIF-90 suppresses the emission of RhB, while the competitive coordination between ATP and the metal node of ZIF-90 dissembles ZIFs, resulting in the release of RhB for ATP sensing. With this method, we are able to image mitochondrial ATP in live cells and study the ATP level fluctuation in cellular glycolysis and apoptosis processes. The strategy reported here could be further extended to tune nanoscale ZIFs inside live cells for targeted delivery of therapeutics to subcellular organelles for advanced biomedical applications.

Block Copolymer Electrolytes: Thermodynamics, Ion Transport, and Use in Solid- State Lithium/Sulfur Cells

Science.gov (United States)

Teran, Alexander Andrew

anode, the compatibility of the sulfur cathode was explored. The sulfur cathode presents many unique challenges, including the generation of soluble lithium polysulfides (Li2Sx, 2 ≤ x ≤ 8) during discharge. The solubility of such species in block copolymers and their effect on morphology was examined. The lithium polysulfides were found to exhibit similar solubility in the block copolymers as in typical organic electrolytes, however induced unusual and unexpected phase behavior in the block copolymers. Inspired by successful efforts to physically confine the soluble lithium polysulfides via nanostructured carbon-sulfur composites in the cathode, our nanostructured block copolymer electrolytes were employed in full electrochemical cells with a lithium metal anode and sulfur cathode. Different cathode compositions, electrolyte additives, and cell architectures were tested. Surprisingly, the polysulfides diffused readily from the cathode through the block copolymer electrolyte, and the normally robust SEO|Li metal interface was detrimentally affected their presence during cycling. The polysulfides appeared to change the mechanical properties of the electrolyte such that intimate contact with the lithium metal was lost. Several promising strategies to overcome this problem were investigated and offer exciting avenues for improvement for future researchers. (Abstract shortened by UMI.).

miR-204 inhibits angiogenesis and promotes sensitivity to cetuximab in head and neck squamous cell carcinoma cells by blocking JAK2-STAT3 signaling.

Science.gov (United States)

Wu, Qingwei; Zhao, Yingying; Wang, Peihua

2018-03-01

This study aims to investigate the roles of miR-204 in tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). Here, we found that miR-204 level was reduced in HNSCC tissues relative to that in normal adjacent tissues. Overexpression of miR-204 promoted tumor angiogenesis in HNSCC cells. Mechanistically, JAK2 was identified as a direct target of miR-204, and miR-204 overexpression blocked JAK2/STAT3 pathway. Moreover, overexpression of JAK2 attenuated the inhibition of miR-204 on tumor angiogenesis of HNSCC. Furthermore, overexpression of miR-204 enhanced sensitivity of cetuximab in HNSCC cells, this effect was attenuated by JAK2 overexpression too. Importantly, JAK2 expression was negatively correlated with miR-204 level in HNSCC tissues. Therefore, miR-204 acts as a tumor suppressor by blocking JAK2/STAT3 pathway in HNSCC cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

Science.gov (United States)

Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

2002-01-01

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

Science.gov (United States)

Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

2016-01-01

Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

Science.gov (United States)

Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

2017-03-27

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Central dogma at the single-molecule level in living cells.

Science.gov (United States)

Li, Gene-Wei; Xie, X Sunney

2011-07-20

Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

Science.gov (United States)

Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

2016-01-01

The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

A combined optical and atomic force microscope for live cell investigations

International Nuclear Information System (INIS)

Madl, Josef; Rhode, Sebastian; Stangl, Herbert; Stockinger, Hannes; Hinterdorfer, Peter; Schuetz, Gerhard J.; Kada, Gerald

2006-01-01

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ('receptor-positive sites') was significantly higher than that on sites lacking receptors

Raman microscopy of individual living human embryonic stem cells

Science.gov (United States)

Novikov, S. M.; Beermann, J.; Bozhevolnyi, S. I.; Harkness, L. M.; Kassem, M.

2010-04-01

We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.

Raman spectroscopy for grading of live osteosarcoma cells.

Science.gov (United States)

Chiang, Yi-Hung; Wu, Stewart H; Kuo, Yi-Chun; Chen, How-Foo; Chiou, Arthur; Lee, Oscar K

2015-04-18

Osteosarcoma is the most common primary malignant bone tumor, and the grading of osteosarcoma cells relies on traditional histopathology and molecular biology methods, which require RNA extraction, protein isolation and immunohistological staining. All these methods require cell isolation, lysis or fixation, which is time-consuming and requires certain amount of tumor specimen. In this study, we report the use of Raman spectroscopy for grading of malignant osteosarcoma cells. We demonstrate that, based on the detection of differential production of mineral species, Raman spectroscopy can be used as a live cell analyzer to accurately assess the grades of osteosarcoma cells by evaluating their mineralization levels. Mineralization level was assessed by measuring amount of hydroxyapatite (HA), which is highly expressed in mature osteoblasts, but not in poorly differentiated osteosarcoma cell or mesenchymal stem cells, the putative cell-of-origin of osteosarcoma. We found that under Raman spectroscopy, the level of HA production was high in MG-63 cells, which are low-grade. Moreover, hydroxyapatite production was low in high-grade osteosarcoma cells such as 143B and SaOS2 cells (p Raman spectroscopy for the measurement of HA production by the protocol reported in this study may serve as a useful tool to rapidly and accurately assess the degree of malignancy in osteosarcoma cells in a label-free manner. Such application may shorten the period of pathological diagnosis and may benefit patients who are inflicted with osteosarcoma.

Lives of a Cell: 40 Years Later, A Third Interpretation

Centers for Disease Control (CDC) Podcasts

2015-06-16

Reginald Tucker reads an abridged version of the article Lives of a Cell: 40 Years Later, A Third Interpretation.  Created: 6/16/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 6/18/2015.

Live cell CRISPR-imaging in plants reveals dynamic telomere movements

KAUST Repository

Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F.; Puchta, Holger; Houben, Andreas

2017-01-01

of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking

Magnetogenetic control of protein gradients inside living cells with high spatial and temporal resolution.

Science.gov (United States)

Etoc, Fred; Vicario, Chiara; Lisse, Domenik; Siaugue, Jean-Michel; Piehler, Jacob; Coppey, Mathieu; Dahan, Maxime

2015-05-13

Tools for controlling the spatial organization of proteins are a major prerequisite for deciphering mechanisms governing the dynamic architecture of living cells. Here, we have developed a generic approach for inducing and maintaining protein gradients inside living cells by means of biofunctionalized magnetic nanoparticles (MNPs). For this purpose, we tailored the size and surface properties of MNPs in order to ensure unhindered mobility in the cytosol. These MNPs with a core diameter below 50 nm could be rapidly relocalized in living cells by exploiting biased diffusion at weak magnetic forces in the femto-Newton range. In combination with MNP surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, we here present a comprehensive technology for controlling intracellular protein gradients with a temporal resolution of a few tens of seconds.

A Novel Technique to Follow Consequences of Exogenous Factors, Including Therapeutic Drugs, on Living Human Breast Epithelial Cells

Science.gov (United States)

1999-07-01

and lipid vectors, are being tested. Concurrent with the development of procedures for live - cell imaging , we are examining the distribution of proteins...dimensional matrix. These studies have not yet begun. There are a number of procedures that must be developed and perfected in the live - cell imaging , as...components of the Wnt signaling pathway are too preliminary and require additional research prior to publication. (9) CONCLUSIONS Live cell imaging of

Engineering Multifunctional Living Paints: Thin, Convectively-Assembled Biocomposite Coatings of Live Cells and Colloidal Latex Particles Deposited by Continuous Convective-Sedimentation Assembly

Science.gov (United States)

Jenkins, Jessica Shawn

Advanced composite materials could be revolutionized by the development of methods to incorporate living cells into functional materials and devices. This could be accomplished by continuously and rapidly depositing thin ordered arrays of adhesive colloidal latex particles and live cells that maintain stability and preserve microbial reactivity. Convective assembly is one method of rapidly assembling colloidal particles into thin (advantages over thicker randomly ordered composites, including enhanced cell stability and increased reactivity through minimized diffusion resistance to nutrients and reduced light scattering. This method can be used to precisely deposit live bacteria, cyanobacteria, yeast, and algae into biocomposite coatings, forming reactive biosensors, photoabsorbers, or advanced biocatalysts. This dissertation developed new continuous deposition and coating characterization methods for fabricating and characterizing 90 hours) photohydrogen production under anoxygenic conditions. Nutrient reduction slows cell division, minimizing coating outgrowth, and promotes photohydrogen generation, improving coating reactivity. Scanning electron microscopy of microstructure revealed how coating reactivity can be controlled by the size and distribution of the nanopores in the biocomposite layers. Variations in colloid microsphere size and suspension composition do not affect coating reactivity, but both parameters alter coating microstructure. Porous paper coated with thin coatings of colloidal particles and cells to enable coatings to be used in a gas-phase without dehydration may offer higher volumetric productivity for hydrogen production. Future work should focus on optimization of cell density, light intensity, media cycling, and acetate concentration.

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

Science.gov (United States)

Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

2013-01-01

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

324 and 325 Building Hot Cell Cleanout Program: Air lock cover block refurbishment

International Nuclear Information System (INIS)

Katayama, Y.B.; Holton, L.K. Jr.; Gale, R.M.

1989-05-01

The high-density concrete cover blocks shielding the pipe trench in the hot-cell air lock of the 324 Building Radiochemical Engineering Cells had accumulated fixed radioactivity ranging from 1100 to 22, 000 mrad/hr. A corresponding increase in the radiation exposure to personnel entering the air lock, together with ALARA concerns, led to the removal of the contaminated concrete surface with a hydraulic spaller and the emplacement of a stainless steel covering over a layer of grout. The resultant saving in radiation exposure is estimated to be 7200 mrad for personnel completing burial box runs for the 324 and 325 Building Hot Cell Cleanout Program. Radiation exposure to all staff members entering the air lock is now at least 50% lower. 3 refs., 22 figs., 1 tab

Hemi-fused structure mediates and controls fusion and fission in live cells.

Science.gov (United States)

Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

2016-06-23

Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

Diagnostic utility of the cell block method versus the conventional smear study in pleural fluid cytology.

Science.gov (United States)

Shivakumarswamy, Udasimath; Arakeri, Surekha U; Karigowdar, Mahesh H; Yelikar, Br

2012-01-01

The cytological examinations of serous effusions have been well-accepted, and a positive diagnosis is often considered as a definitive diagnosis. It helps in staging, prognosis and management of the patients in malignancies and also gives information about various inflammatory and non-inflammatory lesions. Diagnostic problems arise in everyday practice to differentiate reactive atypical mesothelial cells and malignant cells by the routine conventional smear (CS) method. To compare the morphological features of the CS method with those of the cell block (CB) method and also to assess the utility and sensitivity of the CB method in the cytodiagnosis of pleural effusions. The study was conducted in the cytology section of the Department of Pathology. Sixty pleural fluid samples were subjected to diagnostic evaluation for over a period of 20 months. Along with the conventional smears, cell blocks were prepared by using 10% alcohol-formalin as a fixative agent. Statistical analysis with the 'z test' was performed to identify the cellularity, using the CS and CB methods. Mc. Naemer's χ(2)test was used to identify the additional yield for malignancy by the CB method. Cellularity and additional yield for malignancy was 15% more by the CB method. The CB method provides high cellularity, better architectural patterns, morphological features and an additional yield of malignant cells, and thereby, increases the sensitivity of the cytodiagnosis when compared with the CS method.

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays.

Science.gov (United States)

Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D; Chorro, Laurent; Carlin, Leo M; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S

2013-02-19

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

In situ and real-time small-angle neutron scattering studies of living anionic polymerization process and polymerization-induced self-assembly of block copolymers

International Nuclear Information System (INIS)

Tanaka, H.; Yamauchi, K.; Hasegawa, H.; Miyamoto, N.; Koizumi, S.; Hashimoto, T.

2006-01-01

We have studied a simultaneous living anionic polymerization process of isoprene and deuterated styrene in deuterated benzene with sec-buthyl lithium as an initiator into polyisoprene-block-poly(styrene-d 8 ) and the polymerization-induced self-assembling process. This polymerization-induced self-assembling process was directly observed by an in situ and real-time small-angle neutron scattering (SANS) experiment. The time-resolved SANS studies enabled us to explore a time evolution of hierarchical structures induced by a time evolution of the primary structure (linear sequential connection of two monomers)

Effect of infrared light on live blood cells: Role of β-carotene.

Science.gov (United States)

Barkur, Surekha; Bankapur, Aseefhali; Chidangil, Santhosh; Mathur, Deepak

2017-06-01

We have utilized Raman tweezers to measure and assign micro-Raman spectra of optically trapped, live red blood cells (RBCs), white blood cells (WBCs) and platelets. Various types of WBCs- both granulocytes, lymphocytes, and their different types have been studied. The Raman bands are assigned to different biomolecules of blood cells. The Raman spectra thus obtained has been enabled detection of β-carotene in these blood cells, the spectral features of which act as a signature that facilitates experimental probing of the effect of 785nm laser light on different blood cells as a function of incident laser power in the mW range. The spectral changes that we obtain upon laser irradiation indicate that, both haemoglobin as well as the cell membrane sustains damage. In case of lymphocytes and platelets the peaks corresponding to β-carotene showed drastic changes. Thorough analysis of the spectral changes indicates possibility of free radical induced damage of β-carotene in lymphocytes and platelets. Among different blood cells, RBCs have a power threshold of only 10mW. The power threshold for other types of blood cells is somewhat higher, but always below about 30mW. These values are likely to serve as useful guides for Raman tweezers based experiments on live cells. Copyright © 2017. Published by Elsevier B.V.

Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells.

Science.gov (United States)

Okuno, Masanari; Hamaguchi, Hiro-o

2010-12-15

We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

Nanochannel Electroporation as a Platform for Living Cell Interrogation in Acute Myeloid Leukemia.

Science.gov (United States)

Zhao, Xi; Huang, Xiaomeng; Wang, Xinmei; Wu, Yun; Eisfeld, Ann-Kathrin; Schwind, Sebastian; Gallego-Perez, Daniel; Boukany, Pouyan E; Marcucci, Guido I; Lee, Ly James

2015-12-01

A living cell interrogation platform based on nanochannel electroporation is demonstrated with analysis of RNAs in single cells. This minimally invasive process is based on individual cells and allows both multi-target analysis and stimulus-response analysis by sequential deliveries. The unique platform possesses a great potential to the comprehensive and lysis-free nucleic acid analysis on rare or hard-to-transfect cells.

Labeling proteins on live mammalian cells using click chemistry.

Science.gov (United States)

Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

2015-05-01

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

Very thin thermally stable TiO2 blocking layers with enhanced electron transfer for solar cells

Czech Academy of Sciences Publication Activity Database

Kment, Š.; Krýsová, Hana; Hubička, Zdeněk; Kmentová, H.; Kavan, Ladislav; Zbořil, R.

2017-01-01

Roč. 9, DEC 2017 (2017), s. 122-129 ISSN 2352-9407 R&D Projects: GA MŠk(CZ) LM2015073; GA ČR GA13-07724S Grant - others:GA MŠk(CZ) LO1305 Institutional support: RVO:61388955 ; RVO:68378271 Keywords : Cyclic voltammetry * Impedance spectroscopy * Photochemistry * Solar cell * TiO blocking layer 2 Subject RIV: CG - Electrochemistry; BM - Solid Matter Physics ; Magnetism (FZU-D) OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis); Condensed matter physics (including formerly solid state physics, supercond.) (FZU-D)

In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells.

Science.gov (United States)

Espino, Jessica A; Mali, Vishaal S; Jones, Lisa M

2015-08-04

Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.

DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

Science.gov (United States)

Jing, Chaoran; Cornish, Virginia W.

2013-01-01

Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

A combined optical and atomic force microscope for live cell investigations

Energy Technology Data Exchange (ETDEWEB)

Madl, Josef [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Rhode, Sebastian [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Stangl, Herbert [Institute for Medical Chemistry, Medical University Vienna, Waehringerstr. 10, 1090 Vienna (Austria); Stockinger, Hannes [Department of Molecular Immunology, Center for Biomolecular Medicine and Pharmacology, Medical University Vienna, Lazarettgasse 19, 1090 Vienna (Austria); Hinterdorfer, Peter [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Schuetz, Gerhard J. [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Kada, Gerald [Scientec, Mitterbauerweg 4, 4020 Linz (Austria)]. E-mail: gerald_kada@agilent.com

2006-06-15

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ('receptor-positive sites') was significantly higher than that on sites lacking receptors.

A nucleic acid dependent chemical photocatalysis in live human cells

DEFF Research Database (Denmark)

Arian, Dumitru; Cló, Emiliano; Gothelf, Kurt V

2010-01-01

Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment...

Applications of chitosan-based thermo-sensitive copolymers for harvesting living cell sheet

International Nuclear Information System (INIS)

Chen, J.-P.; Yang, T.-F.

2008-01-01

A thermo-sensitive chitosan-based copolymer hydrogel was used for harvesting living cell sheets. The hydrogel was tested for harvesting 3T3 cells after carrying out cell culture at 37 deg. C and incubating the confluent cells at 20 deg. C for spontaneous detachment of cell sheets from hydrogel surface without enzyme treatment. Results from cell viability assay and microscopy observations demonstrated that cells could attach to the hydrogel surface and maintain high viability and proliferation ability. Cell detachment efficiency from the hydrogel was about 80%. The detached cell sheet retained high viability and could proliferate again after transferred to a new culture surface

Quantitative control of mitochondria transfer between live single cells using a microfluidic device

Directory of Open Access Journals (Sweden)

Ken-Ichi Wada

2017-12-01

Full Text Available Quantitative control of mitochondria transfer between live cells is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA because single mitochondrion transfer to a mtDNA-less (ρ0 cell potentially leads to homoplasmy of mtDNA. In this paper, we describe a method for quantitative control of mitochondria transfer between live single cells. For this purpose, we fabricated novel microfluidic devices having cell paring structures with a 4.1, 5.6 or 10.0 μm-length microtunnel. When cells were fused through a microtunnel using the Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel. Elongation of the cytoplasmic connection led to a decrease in mitochondria transfer to the fusion partner. Moreover, some cell pairs that fused through a 10.0 μm-length microtunnel showed single mitochondrion transfer. Fused cells were spontaneously disconnected from each other when they were recovered in a normal culture medium. These results suggest that our cell fusion method can perform quantitative control of mitochondria transfer that includes a single mitochondrion transfer.

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

Science.gov (United States)

Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N

2010-05-01

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

A device for real-time live-cell microscopy during dynamic dual-modal mechanostimulation

Science.gov (United States)

Lorusso, D.; Nikolov, H. N.; Chmiel, T.; Beach, R. J.; Sims, S. M.; Dixon, S. J.; Holdsworth, D. W.

2017-03-01

Mechanotransduction - the process by which cells sense and respond to mechanical stimuli - is essential for several physiological processes including skeletal homeostasis. Mammalian cells are thought to be sensitive to different modes of mechanical stimuli, including vibration and fluid shear. To better understand the mechanisms underlying the early stages of mechanotransduction, we describe the development of devices for mechanostimulation (by vibration and fluid shear) of live cells that can be integrated with real-time optical microscopy. The integrated system can deliver up to 3 Pa of fluid shear simultaneous with high-frequency sinusoidal vibrations up to 1 g. Stimuli can be applied simultaneously or independently to cells during real-time microscopic imaging. A custom microfluidic chamber was prepared from polydimethylsiloxane on a glass-bottom cell culture dish. Fluid flow was applied with a syringe pump to induce shear stress. This device is compatible with a custom-designed motion control vibration system. A voice coil actuates the system that is suspended on linear air bushings. Accelerations produced by the system were monitored with an on-board accelerometer. Displacement was validated optically using particle tracking digital high-speed imaging (1200 frames per second). During operation at nominally 45 Hz and 0.3 g, displacements were observed to be within 3.56% of the expected value. MC3T3-E1 osteoblast like cells were seeded into the microfluidic device and loaded with the calcium sensitive fluorescent probe fura-2, then mounted onto the dual-modal mechanostimulation platform. Cells were then imaged and monitored for fluorescence emission. In summary, we have developed a system to deliver physiologically relevant vibrations and fluid shear to live cells during real-time imaging and photometry. Monitoring the behavior of live cells loaded with appropriate fluorescent probes will enable characterization of the signals activated during the initial

Primary splenic angiosarcoma with liver metastasis: A rare neoplasm diagnosed on fine-needle aspiration cytology and cell block immunocytochemistry

Directory of Open Access Journals (Sweden)

Saniya Sharma

2018-01-01

Full Text Available Primary splenic angiosarcoma is a rare malignant vascular neoplasm of mesenchymal origin. The tumor is highly aggressive and has a high metastatic potential. It is usually diagnosed on histopathological examination of splenectomy specimen. Only few cases of angiosarcoma diagnosed by fine-needle aspiration (FNA cytology alone have been reported in the literature. The cytologic features of angiosarcoma are heterogeneous, however, diagnosis can be suggested by FNA when vasoformative features are present. A 55-year-old female presented with abdominal pain and hepatosplenomegaly. Computed tomography scan revealed a heterogeneous splenic lesion with liver metastases. FNA from the splenic and liver lesions showed moderately pleomorphic tumor cells closely associated with anastomosing vascular channels. Cell block immunocytochemistry (ICC showed tumor cells positive for CD31, CD34, CD68 as well as for CD99. FNA supplemented by cell block ICC can render a definite diagnosis of primary splenic angiosarcoma with liver metastasis.

Rapid relief of block by mecamylamine of neuronal nicotinic acetylcholine receptors of rat chromaffin cells in vitro: an electrophysiological and modeling study.

Science.gov (United States)

Giniatullin, R A; Sokolova, E M; Di Angelantonio, S; Skorinkin, A; Talantova, M V; Nistri, A

2000-10-01

The mechanism responsible for the blocking action of mecamylamine on neuronal nicotinic acetylcholine receptors (nAChRs) was studied on rat isolated chromaffin cells recorded under whole-cell patch clamp. Mecamylamine strongly depressed (IC(50) = 0.34 microM) inward currents elicited by short pulses of nicotine, an effect slowly reversible on wash. The mecamylamine block was voltage-dependent and promptly relieved by a protocol combining membrane depolarization with a nicotine pulse. Either depolarization or nicotine pulses were insufficient per se to elicit block relief. Block relief was transient; response depression returned in a use-dependent manner. Exposure to mecamylamine failed to block nAChRs if they were not activated by nicotine or if they were activated at positive membrane potentials. These data suggest that mecamylamine could not interact with receptors either at rest or at depolarized level. Other nicotinic antagonists like dihydro-beta-erythroidine or tubocurarine did not share this action of mecamylamine although proadifen partly mimicked it. Mecamylamine is suggested to penetrate and block open nAChRs that would subsequently close and trap this antagonist. Computer modeling indicated that the mechanism of mecamylamine blocking action could be described by assuming that 1) mecamylamine-blocked receptors possessed a much slower, voltage-dependent isomerization rate, 2) the rate constant for mecamylamine unbinding was large and poorly voltage dependent. Hence, channel reopening plus depolarization allowed mecamylamine escape and block relief. In the presence of mecamylamine, therefore, nAChRs acquire the new property of operating as coincidence detectors for concomitant changes in membrane potential and receptor occupancy.

Determination of Peroxisomal pH in Living Mammalian Cells Using pHRed.

Science.gov (United States)

Godinho, Luis F; Schrader, Michael

2017-01-01

Organelle pH homeostasis is crucial for maintaining proper cellular function. The nature of the peroxisomal pH remains somewhat controversial, with several studies reporting conflicting results. Here, we describe in detail a rapid and accurate method for the measurement of peroxisomal pH, using the pHRed sensor protein and confocal microscopy of living mammalian cells. pHRed, a ratiometric sensor of pH, is targeted to the peroxisomes by virtue of a C-terminal targeting sequence. The probe has a maximum fluorescence emission at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm, allowing for ratiometric imaging and determination of intracellular pH in live cell microscopy.

Fluorescent tags of protein function in living cells.

Science.gov (United States)

Whitaker, M

2000-02-01

A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection. BioEssays 22:180-187, 2000. Copyright 2000 John Wiley & Sons, Inc.

Automated analysis of invadopodia dynamics in live cells

Directory of Open Access Journals (Sweden)

Matthew E. Berginski

2014-07-01

Full Text Available Multiple cell types form specialized protein complexes that are used by the cell to actively degrade the surrounding extracellular matrix. These structures are called podosomes or invadopodia and collectively referred to as invadosomes. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner.

Nanomembrane-Based, Thermal-Transport Biosensor for Living Cells

KAUST Repository

Elafandy, Rami T.; AbuElela, Ayman; Mishra, Pawan; Janjua, Bilal; Oubei, Hassan M.; Buttner, Ulrich; Majid, Mohammed Abdul; Ng, Tien Khee; Merzaban, Jasmeen; Ooi, Boon S.

2016-01-01

Knowledge of materials' thermal-transport properties, conductivity and diffusivity, is crucial for several applications within areas of biology, material science and engineering. Specifically, a microsized, flexible, biologically integrated thermal transport sensor is beneficial to a plethora of applications, ranging across plants physiological ecology and thermal imaging and treatment of cancerous cells, to thermal dissipation in flexible semiconductors and thermoelectrics. Living cells pose extra challenges, due to their small volumes and irregular curvilinear shapes. Here a novel approach of simultaneously measuring thermal conductivity and diffusivity of different materials and its applicability to single cells is demonstrated. This technique is based on increasing phonon-boundary-scattering rate in nanomembranes, having extremely low flexural rigidities, to induce a considerable spectral dependence of the bandgap-emission over excitation-laser intensity. It is demonstrated that once in contact with organic or inorganic materials, the nanomembranes' emission spectrally shift based on the material's thermal diffusivity and conductivity. This NM-based technique is further applied to differentiate between different types and subtypes of cancer cells, based on their thermal-transport properties. It is anticipated that this novel technique to enable an efficient single-cell thermal targeting, allow better modeling of cellular thermal distribution and enable novel diagnostic techniques based on variations of single-cell thermal-transport properties.

Nanomembrane-Based, Thermal-Transport Biosensor for Living Cells

KAUST Repository

Elafandy, Rami T.

2016-11-23

Knowledge of materials\\' thermal-transport properties, conductivity and diffusivity, is crucial for several applications within areas of biology, material science and engineering. Specifically, a microsized, flexible, biologically integrated thermal transport sensor is beneficial to a plethora of applications, ranging across plants physiological ecology and thermal imaging and treatment of cancerous cells, to thermal dissipation in flexible semiconductors and thermoelectrics. Living cells pose extra challenges, due to their small volumes and irregular curvilinear shapes. Here a novel approach of simultaneously measuring thermal conductivity and diffusivity of different materials and its applicability to single cells is demonstrated. This technique is based on increasing phonon-boundary-scattering rate in nanomembranes, having extremely low flexural rigidities, to induce a considerable spectral dependence of the bandgap-emission over excitation-laser intensity. It is demonstrated that once in contact with organic or inorganic materials, the nanomembranes\\' emission spectrally shift based on the material\\'s thermal diffusivity and conductivity. This NM-based technique is further applied to differentiate between different types and subtypes of cancer cells, based on their thermal-transport properties. It is anticipated that this novel technique to enable an efficient single-cell thermal targeting, allow better modeling of cellular thermal distribution and enable novel diagnostic techniques based on variations of single-cell thermal-transport properties.

Influence of different TiO{sub 2} blocking films on the photovoltaic performance of perovskite solar cells

Energy Technology Data Exchange (ETDEWEB)

Zhang, Chenxi; Luo, Yudan; Chen, Xiaohong, E-mail: xhchen@phy.ecnu.edu.cn; Ou-Yang, Wei; Chen, Yiwei; Sun, Zhuo; Huang, Sumei, E-mail: smhuang@phy.ecnu.edu.cn

2016-12-01

Highlights: • TiO{sub 2} blocking layer (BL) was synthesized using various methods. • Effect of BL characteristics on performance of perovskite solar cell was studied. • Charge transfer kinetics of perovskite solar cells with different BLs was explored. • We demonstrated efficient solar cells employing chemical bath deposition based BLs. - Abstract: Organolead trihalide perovskite materials have been successfully used as light absorbers in efficient photovoltaic (PV) cells. Cell structures based on mesoscopic metal oxides and planar heterojunctions have already demonstrated very impressive and brisk advances, holding great potential to grow into a mature PV technology. High power conversion efficiency (PCE) values have been obtained from the mesoscopic configuration in which a few hundred nano-meter thick mesoporous scaffold (e.g. TiO{sub 2} or Al{sub 2}O{sub 3}) infiltrated by perovskite absorber was sandwiched between the electron and hole transport layers. A uniform and compact hole-blocking layer is necessary for high efficient perovskite-based thin film solar cells. In this study, we investigated the characteristics of TiO{sub 2} compact layer using various methods and its effects on the PV performance of perovskite solar cells. TiO{sub 2} compact layer was prepared by a sol-gel method based on titanium isopropoxide and HCl, spin-coating of titanium diisopropoxide bis (acetylacetonate), screen-printing of Dyesol’s bocking layer titania paste, and a chemical bath deposition (CBD) technique via hydrolysis of TiCl{sub 4}, respectively. The morphological and micro-structural properties of the formed compact TiO{sub 2} layers were characterized by scanning electronic microscopy and X-ray diffraction. The analyses of devices performance characteristics showed that surface morphologies of TiO{sub 2} compact films played a critical role in affecting the efficiencies. The nanocrystalline TiO{sub 2} film deposited via the CBD route acts as the most efficient

Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

International Nuclear Information System (INIS)

Liu, Jianbo; Zhang, Pengfei; Yang, Xiaohai; Wang, Kemin; Guo, Qiuping; Huang, Jin; Li, Wei

2014-01-01

Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses. (paper)

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

Science.gov (United States)

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-01-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

Science.gov (United States)

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-03

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Nanograting-based plasmon enhancement for total internal reflection fluorescence microscopy of live cells

International Nuclear Information System (INIS)

Kim, Kyujung; Cho, Eun-Jin; Suh, Jin-Suck; Huh, Yong-Min; Kim, Donghyun; Kim, Dong Jun

2009-01-01

We investigated evanescent field enhancement based on subwavelength nanogratings for improved sensitivity in total internal reflection microscopy of live cells. The field enhancement is associated with subwavelength-grating-coupled plasmon excitation. An optimum sample employed a silver grating on a silver film and an SF10 glass substrate. Field intensity was enhanced by approximately 90% when measured by fluorescent excitation of microbeads relative to that on a bare prism as a control, which is in good agreement with numerical results. The subwavelength-grating-mediated field enhancement was also applied to live cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label.

Science.gov (United States)

Carlson, Alicia L; Fujisaki, Joji; Wu, Juwell; Runnels, Judith M; Turcotte, Raphaël; Spencer, Joel A; Celso, Cristina Lo; Scadden, David T; Strom, Terry B; Lin, Charles P

2013-01-01

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4(+) T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.

Live birth potential of good morphology and vitrified blastocysts presenting abnormal cell divisions

DEFF Research Database (Denmark)

Azzarello, Antonino; Høst, Thomas; Hay-Schmidt, Anders

2017-01-01

a lower live birth rate (17.0%) than blastocyst with solely regular cell divisions (29.3%). ACDs could occur at more than one cell division in the same good morphology blastocyst. Reported as independent events, we observed ACDs occurring more frequently at the later cell cycles (1st: 1.3%; 2nd: 8.0%; 3rd...

Raman tweezers in microfluidic systems for analysis and sorting of living cells

Science.gov (United States)

Pilát, Zdeněk.; Ježek, Jan; Kaňka, Jan; Zemánek, Pavel

2014-12-01

We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment, dedicated to identification and sorting of biological objects, such as living cells of various unicellular organisms. Our main goal was to create a robust and universal platform for non-destructive and non-contact sorting of micro-objects based on their Raman spectral properties. This approach allowed us to collect spectra containing information about the chemical composition of the objects, such as the presence and composition of pigments, lipids, proteins, or nucleic acids, avoiding artificial chemical probes such as fluorescent markers. The non-destructive nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used a mixture of polystyrene micro-particles and algal cells to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.

Cocompartmentation of proteins and K+ within the living cell

International Nuclear Information System (INIS)

Kellermayer, M.; Ludany, A.; Jobst, K.; Szucs, G.; Trombitas, K.; Hazlewood, C.F.

1986-01-01

Monolayer H-50 tissue culture cells were treated with Triton X-100 and Brij 58 nonionic detergents, and their electron microscopic morphology along with the release of the intracellular proteins [ 35 S]methionine-labelled and 42 K-labelled K + were studied. Although Triton X-100 was more effective, both detergents removed the lipoid membranes within 5 min. The mobilization and solubilization of the cytoplasmic and nuclear proteins occurred much faster with Triton X-100 than with Brij 58. In Triton X-100-treated cells, the loss of K + was complete within 2 min. The loss of K + from the Brij 58-treated cells was complete only after 10 min and the mobilization of K + showed sigmoid-type release kinetics. These results support the view that most of K + and diffusible proteins are not freely dissolved in the cellular water, but they are cocompartmentalized inside the living cell

Diagnostic utility of the cell block method versus the conventional smear study in pleural fluid cytology

Directory of Open Access Journals (Sweden)

Udasimath Shivakumarswamy

2012-01-01

Full Text Available Background: The cytological examinations of serous effusions have been well-accepted, and a positive diagnosis is often considered as a definitive diagnosis. It helps in staging, prognosis and management of the patients in malignancies and also gives information about various inflammatory and non-inflammatory lesions. Diagnostic problems arise in everyday practice to differentiate reactive atypical mesothelial cells and malignant cells by the routine conventional smear (CS method. Aims: To compare the morphological features of the CS method with those of the cell block (CB method and also to assess the utility and sensitivity of the CB method in the cytodiagnosis of pleural effusions. Materials and Methods: The study was conducted in the cytology section of the Department of Pathology. Sixty pleural fluid samples were subjected to diagnostic evaluation for over a period of 20 months. Along with the conventional smears, cell blocks were prepared by using 10% alcohol-formalin as a fixative agent. Statistical analysis with the ′z test′ was performed to identify the cellularity, using the CS and CB methods. Mc. Naemer′s χ2 test was used to identify the additional yield for malignancy by the CB method. Results: Cellularity and additional yield for malignancy was 15% more by the CB method. Conclusions: The CB method provides high cellularity, better architectural patterns, morphological features and an additional yield of malignant cells, and thereby, increases the sensitivity of the cytodiagnosis when compared with the CS method.

Influence of the environment and phototoxicity of the live cell imaging system at IMP microbeam facility

Science.gov (United States)

Liu, Wenjing; Du, Guanghua; Guo, Jinlong; Wu, Ruqun; Wei, Junzhe; Chen, Hao; Li, Yaning; Zhao, Jing; Li, Xiaoyue

2017-08-01

To investigate the spatiotemporal dynamics of DNA damage and repair after the ion irradiation, an online live cell imaging system has been established based on the microbeam facility at Institute of Modern Physics (IMP). The system could provide a sterile and physiological environment by making use of heating plate and live cell imaging solution. The phototoxicity was investigated through the evaluation of DNA repair protein XRCC1 foci formed in HT1080-RFP cells during the imaging exposure. The intensity of the foci induced by phototoxicity was much lower compared with that of the foci induced by heavy ion hits. The results showed that although spontaneous foci were formed due to RFP exposure during live cell imaging, they had little impact on the analysis of the recruitment kinetics of XRCC1 in the foci induced by the ion irradiation.

An efficient, block-by-block algorithm for inverting a block tridiagonal, nearly block Toeplitz matrix

International Nuclear Information System (INIS)

Reuter, Matthew G; Hill, Judith C

2012-01-01

We present an algorithm for computing any block of the inverse of a block tridiagonal, nearly block Toeplitz matrix (defined as a block tridiagonal matrix with a small number of deviations from the purely block Toeplitz structure). By exploiting both the block tridiagonal and the nearly block Toeplitz structures, this method scales independently of the total number of blocks in the matrix and linearly with the number of deviations. Numerical studies demonstrate this scaling and the advantages of our method over alternatives.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

Energy Technology Data Exchange (ETDEWEB)

Costantini, Lindsey M. [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Irvin, Susan C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Kennedy, Steven C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Guo, Feng [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Goldstein, Harris; Herold, Betsy C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Snapp, Erik L., E-mail: erik-lee.snapp@einstein.yu.edu [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)

2015-02-15

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

International Nuclear Information System (INIS)

Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

2015-01-01

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells

A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells

Science.gov (United States)

Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying

2017-06-01

As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.

Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC) and the thrombopoietin receptor (Mpl).

Science.gov (United States)

Zheng, Yuan-Bin; Xiao, Ying-Ying; Tan, Peng; Zhang, Qing; Xu, Peilin

2012-01-01

We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC and the thrombopoietin receptor (Mpl.

Directory of Open Access Journals (Sweden)

Yuan-Bin Zheng

Full Text Available We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER, Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami resulted in increased levels of hNUDC or hNUDC(1-159 secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

Aplicabilidade da técnica de cell block na avaliação citológica do conteúdo de lesões de aspecto cístico dos maxilares Applicability of cell block technique for cytological evaluation of biological material collected from maxillary cystic lesions

Directory of Open Access Journals (Sweden)

Grasieli de Oliveira Ramos

2012-04-01

Full Text Available INTRODUÇÃO: A busca por novos métodos que auxiliem e simplifiquem de maneira eficaz o diagnóstico de lesões maxilares, cistos e tumores, objetiva beneficiar os pacientes e facilitar a atuação dos profissionais da área de diagnóstico bucal. Além dos dados clínicos, radiográficos e histopatológicos classicamente utilizados nos protocolos de investigação das lesões maxilares, a adaptação de técnicas já consagradas na medicina pode ser de grande valia. A técnica de cell block se propõe a auxiliar nesse processo, pois consiste na análise citológica de materiais, efusões e líquidos, coletados de lesões passíveis de punção aspirativa, como cistos e tumores císticos dos maxilares. OBJETIVO: Demonstrar a aplicabilidade da técnica de cell block para avaliação citológica de material biológico coletado a partir de lesões císticas dos maxilares. RESULTADOS: Das 20 lesões, das quais o conteúdo foi processado pela técnica, a avaliação citológica de cinco casos de tumores odontogênicos ceratocísticos (TOCs demonstrou a presença predominante de ceratina, sempre com áreas de paraceratina. Nos demais casos (cinco cistos dentígeros, cinco cistos radiculares e cinco cistos residuais foi observada a presença de hemácias, células inflamatórias e fendas de cristais de colesterol. CONCLUSÃO: A avaliação citológica, a partir da técnica de cell block, foi útil no estabelecimento do diagnóstico diferencial entre TOC e demais lesões estudadas, cistos radicular, residual e dentígero.INTRODUCTION: The search for new methods that aid and optimize the diagnosis of cystic and tumoral maxillary lesions aims to benefit both patients and professionals from oral diagnosis. In addition to clinical, radiographic and histological findings traditionally used in research protocol for maxillary lesions, the adaptation of widely used medical techniques may be very helpful. The cell block procedure streamlines this process

Reforming residential electricity tariff in China: Block tariffs pricing approach

International Nuclear Information System (INIS)

Sun, Chuanwang; Lin, Boqiang

2013-01-01

The Chinese households that make up approximately a quarter of world households are facing a residential power tariff reform in which a rising block tariff structure will be implemented, and this tariff mechanism is widely used around the world. The basic principle of the structure is to assign a higher price for higher income consumers with low price elasticity of power demand. To capture the non-linear effects of price and income on elasticities, we set up a translog demand model. The empirical findings indicate that the higher income consumers are less sensitive than those with lower income to price changes. We further put forward three proposals of Chinese residential electricity tariffs. Compared to a flat tariff, the reasonable block tariff structure generates more efficient allocation of cross-subsidies, better incentives for raising the efficiency of electricity usage and reducing emissions from power generation, which also supports the living standards of low income households. - Highlights: • We design a rising block tariff structure of residential electricity in China. • We set up a translog demand model to find the non-linear effects on elasticities. • The higher income groups are less sensitive to price changes. • Block tariff structure generates more efficient allocation of cross-subsidies. • Block tariff structure supports the living standards of low income households

Diagnostic Value of Processing Cytologic Aspirates of Renal Tumors in Agar Cell (Tissue) Blocks

DEFF Research Database (Denmark)

Smedts, F.; Schrik, M.; Horn, T.

2010-01-01

smears were prepared after each aspiration for conventional cytology and the remaining aspirate was processed for the improved agar microbiopsy (AM) method. Conventional cytology slides, AM slides and surgical specimens were diagnosed separately, after which the diagnoses were compared....... Immunohistochemistry was performed as required on the AM sections. Surgical specimens served as the gold standard. Results In 53% of conventional cytologic smears, the cellular yield was sufficient to render a correct diagnosis. In 12% the diagnosis was incorrect, in 21% only a differential diagnosis could be fin......-initiated, and in 14% too few diagnostic cells were present in the conventional smears for cytologic diagnosis. It was, however, possible to correctly diagnose histologic sections from 97% of AM tissue blocks. In 11 cases this was facilitated with immunochemistry. In only 1 case did the AM tissue block contain too few...

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis.

Science.gov (United States)

Agrawal, Smriti M; Silva, Claudia; Wang, Janet; Tong, Jade Pui-Wai; Yong, V Wee

2012-04-05

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis

Directory of Open Access Journals (Sweden)

Agrawal Smriti M

2012-04-01

Full Text Available Abstract Background Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin is an inducer of the expression of several matrix metalloproteinases (MMPs. We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS, experimental autoimmune encephalomyelitis (EAE. Methods To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Results Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9 production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. Conclusions We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

Monitoring of living cell attachment and spreading using reverse symmetry waveguide sensing

DEFF Research Database (Denmark)

Horvath, R.; Pedersen, H.C.; Skivesen, N.

2005-01-01

The effect of the attachment and spreading of living cells on the modes of a grating coupled reverse symmetry waveguide sensor is investigated in real time. The reverse symmetry design has an increased probing depth into the sample making it well suited for the monitoring of cell morphology....... As a result, significant changes in the incoupling peak height and peak shape were observed during cell attachment and spreading. It is suggested that the area under the incoupling peaks reflects the initial cell attachment process, while the mean peak position is mostly governed by the spreading of the cells...

Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

Science.gov (United States)

Surmacki, Jakub M.; Quirós Gonzalez, Isabel; Bohndiek, Sarah E.

2018-02-01

Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (pmicro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

Molecular building blocks and their architecture in biologically/environmentally compatible soft matter chemical machinery.

Science.gov (United States)

Toyota, Taro; Banno, Taisuke; Nitta, Sachiko; Takinoue, Masahiro; Nomoto, Tomonori; Natsume, Yuno; Matsumura, Shuichi; Fujinami, Masanori

2014-01-01

This review briefly summarizes recent developments in the construction of biologically/environmentally compatible chemical machinery composed of soft matter. Since environmental and living systems are open systems, chemical machinery must continuously fulfill its functions not only through the influx and generation of molecules but also via the degradation and dissipation of molecules. If the degradation or dissipation of soft matter molecular building blocks and biomaterial molecules/polymers can be achieved, soft matter particles composed of them can be used to realize chemical machinery such as selfpropelled droplets, drug delivery carriers, tissue regeneration scaffolds, protocell models, cell-/tissuemarkers, and molecular computing systems.

Digital photocontrol of the network of live excitable cells

Science.gov (United States)

Erofeev, I. S.; Magome, N.; Agladze, K. I.

2011-11-01

Recent development of tissue engineering techniques allows creating and maintaining almost indefinitely networks of excitable cells with desired architecture. We coupled the network of live excitable cardiac cells with a common computer by sensitizing them to light, projecting a light pattern on the layer of cells, and monitoring excitation with the aid of fluorescent probes (optical mapping). As a sensitizing substance we used azobenzene trimethylammonium bromide (AzoTAB). This substance undergoes cis-trans-photoisomerization and trans-isomer of AzoTAB inhibits excitation in the cardiac cells, while cis-isomer does not. AzoTAB-mediated sensitization allows, thus, reversible and dynamic control of the excitation waves through the entire cardiomyocyte network either uniformly, or in a preferred spatial pattern. Technically, it was achieved by coupling a common digital projector with a macroview microscope and using computer graphic software for creating the projected pattern of conducting pathways. This approach allows real time interactive photocontrol of the heart tissue.

Developmental block and programmed cell death in Bos indicus embryos: effects of protein supplementation source and developmental kinetics.

Directory of Open Access Journals (Sweden)

Sheila Merlo Garcia

Full Text Available The aims of this study were to determine if the protein source of the medium influences zebu embryo development and if developmental kinetics, developmental block and programmed cell death are related. The culture medium was supplemented with either fetal calf serum or bovine serum albumin. The embryos were classified as Fast (n = 1,235 or Slow (n = 485 based on the time required to reach the fourth cell cycle (48 h and 90 h post insemination - hpi -, respectively. The Slow group was further separated into two groups: those presenting exactly 4 cells at 48 hpi (Slow/4 cells and those that reached the fourth cell cycle at 90 hpi (Slow. Blastocyst quality, DNA fragmentation, mitochondrial membrane potential and signs of apoptosis or necrosis were evaluated. The Slow group had higher incidence of developmental block than the Fast group. The embryos supplemented with fetal calf serum had lower quality. DNA fragmentation and mitochondrial membrane potential were absent in embryos at 48 hpi but present at 90 hpi. Early signs of apoptosis were more frequent in the Slow and Slow/4 cell groups than in the Fast group. We concluded that fetal calf serum reduces blastocyst development and quality, but the mechanism appears to be independent of DNA fragmentation. The apoptotic cells detected at 48 hpi reveal a possible mechanism of programmed cell death activation prior to genome activation. The apoptotic cells observed in the slow-developing embryos suggested a relationship between programmed cell death and embryonic developmental kinetics in zebu in vitro-produced embryos.

Block copolymer directed synthesis of mesoporous TiO 2 for dye-sensitized solar cells

KAUST Repository

Nedelcu, Mihaela

2009-01-01

The morphology of TiO2 plays an important role in the operation of solid-state dye-sensitized solar cells. By using polyisoprene-block- ethyleneoxide (PI-b-PEO) copolymers as structure directing agents for a sol-gel based synthesis of mesoporous TiO2, we demonstrate a strategy for the detailed control of the semiconductor morphology on the 10 nm length scale. The careful adjustment of polymer molecular weight and titania precursor content is used to systematically vary the material structure and its influence upon solar cell performance is investigated. Furthermore, the use of a partially sp 2 hybridized structure directing polymer enables the crystallization of porous TiO2 networks at high temperatures without pore collapse, improving its performance in solid-state dye-sensitized solar cells. © 2009 The Royal Society of Chemistry.

High content live cell imaging for the discovery of new antimalarial marine natural products

Directory of Open Access Journals (Sweden)

Cervantes Serena

2012-01-01

Full Text Available Abstract Background The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum. Methods A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope. Results Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts. Conclusion Collectively, our results show that high-content live cell-imaging (HCLCI can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials.

High content live cell imaging for the discovery of new antimalarial marine natural products.

Science.gov (United States)

Cervantes, Serena; Stout, Paige E; Prudhomme, Jacques; Engel, Sebastian; Bruton, Matthew; Cervantes, Michael; Carter, David; Tae-Chang, Young; Hay, Mark E; Aalbersberg, William; Kubanek, Julia; Le Roch, Karine G

2012-01-03

The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum. A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope. Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts. Collectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials. © 2011 Cervantes et al; licensee BioMed Central Ltd.

Fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells.

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Laetitia Kurzawa

Full Text Available Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.

Block to influenza virus replication in cells preirradiated with ultraviolet light

International Nuclear Information System (INIS)

Mahy, B.W.J.; Carroll, A.R.; Brownson, J.M.T.; McGeoch, D.J.

1977-01-01

Ultraviolet (uv) irradiation of CEF cells immediately before infection with influenza A (fowl plague) virus inhibited virus growth; no inhibition of the growth of a parainfluenza virus (Newcastle disease virus) could be detected in irradiated cells. The kinetics of inhibition after various doses of uv irradiation were multihit, with an extrapolation number of two. When irradiated cells were allowed to photoreactivate by exposure to visible light for 16 hr their capacity to support influenza virus replication was largely restored; this process was sensitive to caffeine, suggesting that it required DNA repair. In CEF cells exposed to 360 ergs/mm 2 of uv radiation the rate of synthesis of host cellular RNA was reduced by more than 90%, and that of host cellular protein by 40 to 50%, as judged by incorporation of precursor molecules into an acid-insoluble form. When such irradiated cells were infected with influenza virus all the genome RNA segments were transcribed, but the overall concentration of virus-specific poly(A)-containing cRNA was reduced about 50-fold. Within this population of cRNA molecules, the RNAs coding for late proteins (HA, NA, and M) were reduced in amount relative to the other segments. The rates of synthesis of the M and HA proteins were specifically reduced in uv-irradiated cells, but the rates of synthesis of the P, NP, and NS proteins were only slightly reduced compared to normal cells. Immunofluorescent studies showed that, in uv-irradiated cells, NP migrated into the nucleus early after infection and later migrated out into the cytoplasm, as in normal cells. In contrast to normal cells, no specific immunofluorescence associated with M protein could be observed in uv-irradiated cells. It is concluded that uv-induced damage to host cellular DNA alters the pattern of RNA transcription in CEF cells infected with influenza virus, and that this results in a block to late protein synthesis which stops virus production

Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays

International Nuclear Information System (INIS)

Noguchi, M.; Yokoya, A.; Narita, A.; Fujii, K.; Kanari, Y.

2015-01-01

Morphological changes in mitochondria induced by X-irradiation in normal murine mammary gland cells were studied with a live-cell microscopic imaging technique. Mitochondria were visualised by staining with a specific fluorescent probe in the cells, which express fluorescent ubiquitination-based cell-cycle indicator 2 (Fucci2) probes to visualise cell cycle. In unirradiated cells, the number of cells with fragmented mitochondria was about 20 % of the total cells through observation period (96 h). In irradiated cells, the population with fragmented mitochondria significantly increased depending on the absorbed dose. Particularly, for 8 Gy irradiation, the accumulation of fragmentation persists even in the cells whose cell cycle came to a stand (80 % in G1 (G0-like) phase). The fraction reached to a maximum at 96 h after irradiation. The kinetics of the fraction with fragmented mitochondria was similar to that for cells in S/G2/M phase (20 %) through the observation period (120 h). The evidences show that, in irradiated cells, some signals are continually released from a nucleus or cytoplasm even in the G0-like cells to operate some sort of protein machineries involved in mitochondrial fission. It is inferred that this delayed mitochondrial fragmentation is strongly related to their dysfunction, and hence might modulate radiobiological effects such as mutation or cell death. (authors)

Live attenuated measles virus vaccine therapy for locally established malignant glioblastoma tumor cells