Lymphocyte migration in the micro-channel of splenic sheathed capillaries in Chinese soft-shelled turtles, Pelodiscus sinensis.

PubMed

Zhang, Qian; Ullah, Shakeeb; Liu, Yi; Yang, Ping; Chen, Bing; Waqas, Yasir; Bao, Huijun; Hu, Lisi; Li, Quanfu; Chen, Qiusheng

2016-01-01

The structural characteristics of the splenic sheathed capillary were investigated using light microscopy and transmission electron microscopy (TEM). This study mainly focused on lymphocyte migration to the splenic white pulp via micro-channels in Chinese soft-shelled turtles, Pelodiscus sinensis. The results showed that the sheathed capillaries in the turtle spleen were high endothelial venule (HEV)-like vessels. These capillaries consist of micro-channels that facilitate lymphocyte migration to the splenic white pulp. The micro-channel is a dynamic structure comprising processes of endothelial cells, supporting cells, and ellipsoid-associated cells (EACs), which provides a microenvironment for lymphocyte migration. The pattern of lymphocyte migration in the micro-channel of the turtle spleen includes the following steps: (i) lymphocyte first adheres to the endothelium of the sheathed capillary, passes through the endothelial cells, and traverses through the basement membrane of the sheathed capillary; (ii) it then enters into the ellipsoid combined with supporting cells and EACs; and (iii) lymphocyte migrates from the ellipsoid to the periellipsoidal lymphatic sheath (PELS) via the micro-channel. This study provides morphological evidence for lymphocyte migration in the micro-channels of turtle spleens and also an insight into the mechanism of lymphocyte homing to the splenic white pulp of reptiles. Copyright © 2015 Elsevier Ltd. All rights reserved.

Retinoids induce differentiation and downregulate telomerase activity and N-Myc to increase sensitivity to flavonoids for apoptosis in human malignant neuroblastoma SH-SY5Y cells.

PubMed

Das, Arabinda; Banik, Naren L; Ray, Swapan K

2009-03-01

Human malignant neuroblastoma is characterized by poor differentiation and uncontrolled proliferation of immature neuroblasts. Retinoids such as all-trans-retinoic acid (ATRA), 13-cis-retinoic acid (13-CRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) at low doses are capable of inducing differentiation, while flavonoids such as (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST) at relatively high dose can induce apoptosis. We used combination of retinoid and flavonoid for controlling growth of malignant neuroblastoma SH-SY5Y cells. Cells were treated with a retinoid (1 microM ATRA, 1 microM 13-CRA, or 0.5 microM 4-HPR) for 7 days and then with a flavonoid (25 microM EGCG or 25 microM GST) for 24 h. Treatment of cells with a low dose of a retinoid for 7 days induced neuronal differentiation with downregulation of telomerase activity and N-Myc but overexpression of neurofilament protein (NFP) and subsequent treatment with a relatively high dose of a flavonoid for 24 h increased apoptosis in the differentiated cells. Besides, retinoids reduced the levels of inflammatory and angiogenic factors. Apoptosis was associated with increases in intracellular free [Ca2+], Bax expression, cytochrome c release from mitochondria and activities of calpain and caspases. Decreases in expression of calpastatin (endogenous calpain inhibitor) and baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins (endogenous caspase inhibitors) favored apoptosis. Treatment of SH-SY5Y cells with EGCG activated caspase-8, indicating induction of the receptor-mediated pathway of apoptosis. Based on our observation, we conclude that combination of a retinoid and a flavonoid worked synergistically for controlling the malignant growth of human neuroblastoma cells.

A three-dimensional hierarchical collagen scaffold fabricated by a combined solid freeform fabrication (SFF) and electrospinning process to enhance mesenchymal stem cell (MSC) proliferation

NASA Astrophysics Data System (ADS)

Ahn, SeungHyun; Koh, Young Ho; Kim, GeunHyung

2010-06-01

Collagen has the advantage of being very similar to macromolecular substances that can be recognized and metabolized in the biological environment. Although the natural material has superior property for this purpose, its use to fabricate reproducible and pore-structure-controlled 3D structures, which are designed to allow the entry of sufficient cells and the easy diffusion of nutrients, has been limited due to its low processability. Here, we propose a hybrid technology that combines a cryogenic plotting system with an electrospinning process. Using this technique, an easily pore-size-controllable hierarchical 3D scaffold consisting of micro-sized highly porous collagen strands and micro/nano-sized collagen fibers was fabricated. The pore structure of the collagen scaffold was controlled by the collagen micro/nanofibers, which were layered in the scaffold. The hierarchical scaffolds were characterized with respect to initial cell attachment and proliferation of bone marrow-derived mesenchymal stem cells within the scaffolds. The hierarchical scaffold exhibited incredibly enhanced initial cell attachment and cell compactness between pores of the plotted scaffold relative to the normally designed 3D collagen scaffold.

Synergistic anticancer effects of combined gamma-tocotrienol and celecoxib treatment are associated with suppression in Akt and NFkappaB signaling.

PubMed

Shirode, Amit B; Sylvester, Paul W

2010-05-01

The selective cyclooxygenase (COX)-2 inhibitor, celecoxib, and the vitamin E isoform, gamma-tocotrienol, both display potent anticancer activity. However, high dose clinical use of selective COX-2 inhibitors has been limited by gastrointestinal and cardiovascular toxicity, whereas limited absorption and transport of gamma-tocotrienol by the body has made it difficult to obtain and sustain therapeutic levels in the blood and target tissues. Studies were conducted to characterize the synergistic anticancer antiproliferative effects of combined low dose celecoxib and gamma-tocotrienol treatment on mammary tumor cells in culture. The highly malignant mouse +SA mammary epithelial cells were maintained in culture on serum-free defined control or treatment media. Treatment effects on COX-1, COX-2, Akt, NFkappaB and prostaglandin E(2) (PGE(2)) synthesis were assessed following a 3- or 4-day culture period. Treatment with 3-4 microM gamma-tocotrienol or 7.5-10 microM celecoxib alone significantly inhibited +SA cell growth in a dose-responsive manner. However, combined treatment with subeffective doses of gamma-tocotrienol (0.25 microM) and celecoxib (2.5 microM) resulted in a synergistic antiproliferative effect, as determined by isobologram analysis, and this growth inhibitory effect was associated with a reduction in PGE(2) synthesis, and decrease in COX-2, phospho-Akt (active), and phospho-NFkappaB (active) levels. These results demonstrate that the synergistic anticancer effects of combined celecoxib and gamma-tocotrienol therapy are mediated by COX-2 dependent and independent mechanisms. These findings also suggest that combination therapy with these agents may provide enhanced therapeutic response in breast cancer patients, while avoiding the toxicity associated with high-dose COX-2 inhibitor monotherapy. 2009 Elsevier Masson SAS. All rights reserved.

Quantifying substrate uptake by individual cells of marine bacterioplankton by catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography.

PubMed

Sintes, Eva; Herndl, Gerhard J

2006-11-01

Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up (3)H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions.

Quantifying Substrate Uptake by Individual Cells of Marine Bacterioplankton by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Microautoradiography▿

PubMed Central

Sintes, Eva; Herndl, Gerhard J.

2006-01-01

Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up 3H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions. PMID:16950912

Image-guided smart laser system for precision implantation of cells in cartilage

NASA Astrophysics Data System (ADS)

Katta, Nitesh; Rector, John A.; Gardner, Michael R.; McElroy, Austin B.; Choy, Kevin C.; Crosby, Cody; Zoldan, Janet; Milner, Thomas E.

2017-03-01

State-of-the-art treatment for joint diseases like osteoarthritis focus on articular cartilage repair/regeneration by stem cell implantation therapy. However, the technique is limited by a lack of precision in the physician's imaging and cell deposition toolkit. We describe a novel combination of high-resolution, rapid scan-rate optical coherence tomography (OCT) alongside a short-pulsed nanosecond thulium (Tm) laser for precise cell seeding in cartilage. The superior beam quality of thulium lasers and wavelength of operation 1940 nm offers high volumetric tissue removal rates and minimizes the residual thermal footprint. OCT imaging enables targeted micro-well placement, precise cell deposition, and feature contrast. A bench-top system is constructed using a 15 W, 1940 nm, nanosecond-pulsed Tm fiber laser (500 μJ pulse energy, 100 ns pulse duration, 30kHz repetition rate) for removing tissue, and a swept source laser (1310 ± 70 nm, 100 kHz sweep rate) for OCT imaging, forming a combined Tm/OCT system - a "smart laser knife". OCT assists the smart laser knife user in characterizing cartilage to inform micro-well placement. The Tm laser creates micro-wells (2.35 mm diameter length, 1.5 mm width, 300 μm deep) and micro-incisions (1 mm wide, 200 μm deep) while OCT image-guidance assists and demonstrates this precision cutting and cell deposition with real-time feedback. To test micro-well creation and cell deposition protocol, gelatin phantoms are constructed mimicking cartilage optical properties and physiological structure. Cell viability is then assessed to illustrate the efficacy of the hydrogel deposition. Automated OCT feedback is demonstrated for cutting procedures to avoid important surface/subsurface structures. This bench-top smart laser knife system described here offers a new image-guided approach to precise stem cell seeding that can enhance the efficacy of articular cartilage repair.

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

PubMed Central

Lipchina, Inna; Elkabetz, Yechiel; Hafner, Markus; Sheridan, Robert; Mihailovic, Aleksandra; Tuschl, Thomas; Sander, Chris; Studer, Lorenz; Betel, Doron

2011-01-01

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area. PMID:22012620

Micro/Nano Structural Tantalum Coating for Enhanced Osteogenic Differentiation of Human Bone Marrow Stem Cells

PubMed Central

Ding, Ding; Xie, Youtao; Li, Kai; Huang, Liping; Zheng, Xuebin

2018-01-01

Recently, tantalum has been attracting much attention for its anticorrosion resistance and biocompatibility, and it has been widely used in surface modification for implant applications. To improve its osteogenic differentiation of human bone marrow stem cells (hBMSCs), a micro/nano structure has been fabricated on the tantalum coating surface through the combination of anodic oxidation and plasma spraying method. The morphology, composition, and microstructure of the modified coating were comprehensively studied by employing scanning electron microscopy (SEM), X-ray diffraction (XRD) as well as transmission electron microscopy (TEM). The effects of hierarchical structures as well as micro-porous structure of tantalum coating on the behavior for human bone marrow stem cells (hBMSCs) were evaluated and compared at both cellular and molecular levels in vitro. The experimental results show that a hierarchical micro/nano structure with Ta2O5 nanotubes spread onto a micro-scale tantalum coating has been fabricated successfully, which is confirmed to promote cell adhesion and spreading. Besides, the hierarchical micro/nano tantalum coating can provide 1.5~2.1 times improvement in gene expression, compared with the micro-porous tantalum coating. It demonstrates that it can effectively enhance the proliferation and differentiation of hBMSCs in vitro. PMID:29614022

Micro/Nano Structural Tantalum Coating for Enhanced Osteogenic Differentiation of Human Bone Marrow Stem Cells.

PubMed

Ding, Ding; Xie, Youtao; Li, Kai; Huang, Liping; Zheng, Xuebin

2018-04-03

Recently, tantalum has been attracting much attention for its anticorrosion resistance and biocompatibility, and it has been widely used in surface modification for implant applications. To improve its osteogenic differentiation of human bone marrow stem cells (hBMSCs), a micro/nano structure has been fabricated on the tantalum coating surface through the combination of anodic oxidation and plasma spraying method. The morphology, composition, and microstructure of the modified coating were comprehensively studied by employing scanning electron microscopy (SEM), X-ray diffraction (XRD) as well as transmission electron microscopy (TEM). The effects of hierarchical structures as well as micro-porous structure of tantalum coating on the behavior for human bone marrow stem cells (hBMSCs) were evaluated and compared at both cellular and molecular levels in vitro. The experimental results show that a hierarchical micro/nano structure with Ta₂O₅ nanotubes spread onto a micro-scale tantalum coating has been fabricated successfully, which is confirmed to promote cell adhesion and spreading. Besides, the hierarchical micro/nano tantalum coating can provide 1.5~2.1 times improvement in gene expression, compared with the micro-porous tantalum coating. It demonstrates that it can effectively enhance the proliferation and differentiation of hBMSCs in vitro.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

PubMed

Flood, P; Alvarez, L; Reynaud, E G

2016-10-11

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Dauer larva quiescence alters the circuitry of microRNA pathways regulating cell fate progression in C. elegans.

PubMed

Karp, Xantha; Ambros, Victor

2012-06-01

In C. elegans larvae, the execution of stage-specific developmental events is controlled by heterochronic genes, which include those encoding a set of transcription factors and the microRNAs that regulate the timing of their expression. Under adverse environmental conditions, developing larvae enter a stress-resistant, quiescent stage called 'dauer'. Dauer larvae are characterized by the arrest of all progenitor cell lineages at a stage equivalent to the end of the second larval stage (L2). If dauer larvae encounter conditions favorable for resumption of reproductive growth, they recover and complete development normally, indicating that post-dauer larvae possess mechanisms to accommodate an indefinite period of interrupted development. For cells to progress to L3 cell fate, the transcription factor Hunchback-like-1 (HBL-1) must be downregulated. Here, we describe a quiescence-induced shift in the repertoire of microRNAs that regulate HBL-1. During continuous development, HBL-1 downregulation (and consequent cell fate progression) relies chiefly on three let-7 family microRNAs, whereas after quiescence, HBL-1 is downregulated primarily by the lin-4 microRNA in combination with an altered set of let-7 family microRNAs. We propose that this shift in microRNA regulation of HBL-1 expression involves an enhancement of the activity of lin-4 and let-7 microRNAs by miRISC modulatory proteins, including NHL-2 and LIN-46. These results illustrate how the employment of alternative genetic regulatory pathways can provide for the robust progression of progenitor cell fates in the face of temporary developmental quiescence.

Panaxadiol, a purified ginseng component, enhances the anti-cancer effects of 5-fluorouracil in human colorectal cancer cells.

PubMed

Li, Xiao-Li; Wang, Chong-Zhi; Mehendale, Sangeeta R; Sun, Shi; Wang, Qi; Yuan, Chun-Su

2009-11-01

Colorectal cancer is a major cause of morbidity and mortality for cancer worldwide. Although 5-fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents in first-line therapy for colorectal cancer, serious side effects limit its clinical usefulness. Panaxadiol (PD) is the purified sapogenin of ginseng saponins, which exhibit anti-tumor activity. In this study, we investigated the possible synergistic anti-cancer effects of PD and 5-FU on a human colorectal cancer cell line, HCT-116. Cell viability was evaluated by an MTS cell proliferation assay. Morphological observation was performed by crystal violet cell viability staining assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or Annexin V/PI. Cell growth was markedly suppressed in HCT-116 cells treated by 5-FU (20-100 microM) for 24 or 48 h with time-dependent effects. The significant suppression on HCT-116 cell proliferation was observed after treatment with PD (25 microM) for 24 and 48 h. Panaxadiol (25 microM) markedly (P < 0.05) enhanced the anti-proliferative effects of 5-FU (5, 10, 20 microM) on HCT-116 cells compared to single treatment of 5-FU for 24 and 48 h. Flow cytometric analysis on DNA indicated that PD and 5-FU selectively arrested cell cycle progression in the G1 phase and S phase (P < 0.01), respectively, compared to the control condition. Combination use of 5-FU with PD significantly (P < 0.001) increased cell cycle arrest in the S phase compared to that treated by 5-FU alone. The combination of 5-FU and PD significantly enhanced the percentage of apoptotic cells when compared with the corresponding cell groups treated by 5-FU alone (P < 0.001). Panaxadiol enhanced the anti-cancer effects of 5-FU on human colorectal cancer cells through the regulation of cell cycle transition and the induction of apoptotic cells.

Hybrid micro/nano-topography of a TiO2 nanotube-coated commercial zirconia femoral knee implant promotes bone cell adhesion in vitro.

PubMed

Frandsen, Christine J; Noh, Kunbae; Brammer, Karla S; Johnston, Gary; Jin, Sungho

2013-07-01

Various approaches have been studied to engineer the implant surface to enhance bone in-growth properties, particularly using micro- and nano-topography. In this study, the behavior of osteoblast (bone) cells was analyzed in response to a titanium oxide (TiO2) nanotube-coated commercial zirconia femoral knee implant consisting of a combined surface structure of a micro-roughened surface with the nanotube coating. The osteoblast cells demonstrated high degrees of adhesion and integration into the surface of the nanotube-coated implant material, indicating preferential cell behavior on this surface when compared to the bare implant. The results of this brief study provide sufficient evidence to encourage future studies. The development of such hierarchical micro- and nano-topographical features, as demonstrated in this work, can provide insightful designs for advanced bone-inducing material coatings on ceramic orthopedic implant surfaces. Copyright © 2013 Elsevier B.V. All rights reserved.

Experimental analysis of performance degradation of micro-tubular solid oxide fuel cells fed by different fuel mixtures

NASA Astrophysics Data System (ADS)

Calise, F.; Restucccia, G.; Sammes, N.

This paper analyzes the thermodynamic and electrochemical dynamic performance of an anode supported micro-tubular solid oxide fuel cell (SOFC) fed by different types of fuel. The micro-tubular SOFC used is anode supported, consisting of a NiO and Gd 0.2Ce 0.8O 2- x (GDC) cermet anode, thin GDC electrolyte, and a La 0.6Sr 0.4Co 0.2Fe 0.8O 3- y (LSCF) and GDC cermet cathode. The fabrication of the cells under investigation is briefly summarized, with emphasis on the innovations with respect to traditional techniques. Such micro-tubular cells were tested using a Test Stand consisting of: a vertical tubular furnace, an electrical load, a galvanostast, a bubbler, gas pipelines, temperature, pressure and flow meters. The tests on the micro-SOFC were performed using H 2, CO, CH 4 and H 2O in different combinations at 550 °C, to determine the cell polarization curves under several load cycles. Long-term experimental tests were also performed in order to assess degradation of the electrochemical performance of the cell. Results of the tests were analyzed aiming at determining the sources of the cell performance degradation. Authors concluded that the cell under investigation is particularly sensitive to the carbon deposition which significantly reduces cell performance, after few cycles, when fed by light hydrocarbons. A significant performance degradation is also detected when hydrogen is used as fuel. In this case, the authors ascribe the degradation to the micro-cracks, the change in materials crystalline structure and problems with electrical connections.

miR-137 Targets p160 Steroid Receptor Coactivators SRC1, SRC2, and SRC3 and Inhibits Cell Proliferation

PubMed Central

Eedunuri, Vijay Kumar; Rajapakshe, Kimal; Fiskus, Warren; Geng, Chuandong; Chew, Sue Anne; Foley, Christopher; Shah, Shrijal S.; Shou, John; Mohamed, Junaith S.; O'Malley, Bert W.

2015-01-01

The p160 family of steroid receptor coactivators (SRCs) are pleiotropic transcription factor coactivators and “master regulators” of gene expression that promote cancer cell proliferation, survival, metabolism, migration, invasion, and metastasis. Cancers with high p160 SRC expression exhibit poor clinical outcomes and resistance to therapy, highlighting the SRCs as critical oncogenic drivers and, thus, therapeutic targets. microRNAs are important epigenetic regulators of protein expression. To examine the regulation of p160 SRCs by microRNAs, we used and combined 4 prediction algorithms to identify microRNAs that could target SRC1, SRC2, and SRC3 expression. For validation of these predictions, we assessed p160 SRC protein expression and cell viability after transfection of corresponding microRNA mimetics in breast cancer, uveal melanoma, and prostate cancer (PC) cell lines. Transfection of selected microRNA mimetics into breast cancer, uveal melanoma, and PC cells depleted SRC protein expression levels and exerted potent antiproliferative activity in these cell types. In particular, microRNA-137 (miR-137) depleted expression of SRC1, SRC2, and very potently, SRC3. The latter effect can be attributed to the presence of 3 miR-137 recognition sequences within the SRC3 3′-untranslated region. Using reverse phase protein array analysis, we identified a network of proteins, in addition to SRC3, that were modulated by miR-137 in PC cells. We also found that miR-137 and its host gene are epigenetically silenced in human cancer specimens and cell lines. These results support the development and testing of microRNA-based therapies (in particular based on restoring miR-137 levels) for targeting the oncogenic family of p160 SRCs in cancer. PMID:26066330

Reprogramming hMSCs morphology with silicon/porous silicon geometric micro-patterns.

PubMed

Ynsa, M D; Dang, Z Y; Manso-Silvan, M; Song, J; Azimi, S; Wu, J F; Liang, H D; Torres-Costa, V; Punzon-Quijorna, E; Breese, M B H; Garcia-Ruiz, J P

2014-04-01

Geometric micro-patterned surfaces of silicon combined with porous silicon (Si/PSi) have been manufactured to study the behaviour of human Mesenchymal Stem Cells (hMSCs). These micro-patterns consist of regular silicon hexagons surrounded by spaced columns of silicon equilateral triangles separated by PSi. The results show that, at an early culture stage, the hMSCs resemble quiescent cells on the central hexagons with centered nuclei and actin/β-catenin and a microtubules network denoting cell adhesion. After 2 days, hMSCs adapted their morphology and cytoskeleton proteins from cell-cell dominant interactions at the center of the hexagonal surface. This was followed by an intermediate zone with some external actin fibres/β-catenin interactions and an outer zone where the dominant interactions are cell-silicon. Cells move into silicon columns to divide, migrate and communicate. Furthermore, results show that Runx2 and vitamin D receptors, both specific transcription factors for skeleton-derived cells, are expressed in cells grown on micropatterned silicon under all observed circumstances. On the other hand, non-phenotypic alterations are under cell growth and migration on Si/PSi substrates. The former consideration strongly supports the use of micro-patterned silicon surfaces to address pending questions about the mechanisms of human bone biogenesis/pathogenesis and the study of bone scaffolds.

Bis(ethyl)norspermine potentiates the apoptotic activity of the pure antiestrogen ICI 182780 in breast cancer cells.

PubMed

Balabhadrapathruni, Srivani; Santhakumaran, Latha M; Thomas, T J; Shirahata, Akira; Gallo, Michael A; Thomas, Thresia

2005-01-01

We studied the effects of ICI 182780 and bis(ethyl)norspermine (BE-3-3-3) on cell growth and apoptosis of estrogen receptor-positive MCF-7 breast cancer cells. Combination treatment with 100 nM ICI 182780 and 5 microM BE-3-3-3 for 6 days inhibited cell growth by 74.3+/-8.4% in MCF-7 cells, compared to that of 25.4+/-5.8 and 45.8+/-12.2%, respectively, when ICI 182780 and BE-3-3-3 were used as single agents. Treatment with 100 nM ICI 182780 and 5 microM BE-3-3-3 as single agents resulted in 9.1+/-1.0% and 35.1+/-4.5% apoptosis, respectively, as measured by APO-BRDU assay. When ICI 182780 and BE-3-3-3 were used in combination, the percentage of apoptosis was 60.6+/-3.8%. Improved efficacy of ICI 182780 and BE-3-3-3 combination on growth inhibition was observed for T-47D cells also. Western blot analysis showed that combinations of ICI 182780 and BE-3-3-3 caused down-regulation of the anti-apoptotic Bcl-2 and Bcl-XL proteins and increased the level of the pro-apoptotic Bax protein. Combination treatment also increased caspase-8 activation. Analysis of polyamine levels 48 h after combination treatment showed that spermidine and spermine levels were down regulated significantly. These studies indicate a potentially effective combination strategy for breast cancer treatment. Our results also link the down-regulation of polyamine pathway to apoptotic cell death and regulation of mediators of cell death.

Macro- and micro-designed chitosan-alginate scaffold architecture by three-dimensional printing and directional freezing.

PubMed

Reed, Stephanie; Lau, Grace; Delattre, Benjamin; Lopez, David Don; Tomsia, Antoni P; Wu, Benjamin M

2016-01-07

While many tissue-engineered constructs aim to treat cartilage defects, most involve chondrocyte or stem cell seeding on scaffolds. The clinical application of cell-based techniques is limited due to the cost of maintaining cellular constructs on the shelf, potential immune response to allogeneic cell lines, and autologous chondrocyte sources requiring biopsy from already diseased or injured, scarce tissue. An acellular scaffold that can induce endogenous influx and homogeneous distribution of native stem cells from bone marrow holds great promise for cartilage regeneration. This study aims to develop such an acellular scaffold using designed, channeled architecture that simultaneously models the native zones of articular cartilage and subchondral bone. Highly porous, hydrophilic chitosan-alginate (Ch-Al) scaffolds were fabricated in three-dimensionally printed (3DP) molds designed to create millimeter scale macro-channels. Different polymer preform casting techniques were employed to produce scaffolds from both negative and positive 3DP molds. Macro-channeled scaffolds improved cell suspension distribution and uptake overly randomly porous scaffolds, with a wicking volumetric flow rate of 445.6 ± 30.3 mm(3) s(-1) for aqueous solutions and 177 ± 16 mm(3) s(-1) for blood. Additionally, directional freezing was applied to Ch-Al scaffolds, resulting in lamellar pores measuring 300 μm and 50 μm on the long and short axes, thus creating micrometer scale micro-channels. After directionally freezing Ch-Al solution cast in 3DP molds, the combined macro- and micro-channeled scaffold architecture enhanced cell suspension uptake beyond either macro- or micro-channels alone, reaching a volumetric flow rate of 1782.1 ± 48 mm(3) s(-1) for aqueous solutions and 440.9 ± 0.5 mm(3) s(-1) for blood. By combining 3DP and directional freezing, we can control the micro- and macro-architecture of Ch-Al to drastically improve cell influx into and distribution within the scaffold, while achieving porous zones that mimic articular cartilage zonal architecture. In future applications, precisely controlled micro- and macro-channels have the potential to assist immediate endogenous bone marrow uptake, stimulate chondrogenesis, and encourage vascularization of bone in an osteochondral scaffold.

Measuring the mechanical properties of plant cells by combining micro-indentation with osmotic treatments.

PubMed

Weber, Alain; Braybrook, Siobhan; Huflejt, Michal; Mosca, Gabriella; Routier-Kierzkowska, Anne-Lise; Smith, Richard S

2015-06-01

Growth in plants results from the interaction between genetic and signalling networks and the mechanical properties of cells and tissues. There has been a recent resurgence in research directed at understanding the mechanical aspects of growth, and their feedback on genetic regulation. This has been driven in part by the development of new micro-indentation techniques to measure the mechanical properties of plant cells in vivo. However, the interpretation of indentation experiments remains a challenge, since the force measures results from a combination of turgor pressure, cell wall stiffness, and cell and indenter geometry. In order to interpret the measurements, an accurate mechanical model of the experiment is required. Here, we used a plant cell system with a simple geometry, Nicotiana tabacum Bright Yellow-2 (BY-2) cells, to examine the sensitivity of micro-indentation to a variety of mechanical and experimental parameters. Using a finite-element mechanical model, we found that, for indentations of a few microns on turgid cells, the measurements were mostly sensitive to turgor pressure and the radius of the cell, and not to the exact indenter shape or elastic properties of the cell wall. By complementing indentation experiments with osmotic experiments to measure the elastic strain in turgid cells, we could fit the model to both turgor pressure and cell wall elasticity. This allowed us to interpret apparent stiffness values in terms of meaningful physical parameters that are relevant for morphogenesis. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastoma cells to lower effective doses of temozolomide in vitro and in a mouse model.

PubMed

Malhotra, Meenakshi; Sekar, Thillai Veerapazham; Ananta, Jeyarama S; Devulapally, Rammohan; Afjei, Rayhaneh; Babikir, Husam A; Paulmurugan, Ramasamy; Massoud, Tarik F

2018-04-20

Temozolomide (TMZ) chemotherapy for glioblastoma (GBM) is generally well tolerated at standard doses but it can cause side effects. GBMs overexpress microRNA-21 and microRNA-10b, two known oncomiRs that promote cancer development, progression and resistance to drug treatment. We hypothesized that systemic injection of antisense microRNAs (antagomiR-21 and antagomiR-10b) encapsulated in cRGD-tagged PEG-PLGA nanoparticles would result in high cellular delivery of intact functional antagomiRs, with consequent efficient therapeutic response and increased sensitivity of GBM cells to lower doses of TMZ. We synthesized both targeted and non-targeted nanoparticles, and characterized them for size, surface charge and encapsulation efficiency of antagomiRs. When using targeted nanoparticles in U87MG and Ln229 GBM cells, we showed higher uptake-associated improvement in sensitivity of these cells to lower concentrations of TMZ in medium. Co-inhibition of microRNA-21 and microRNA-10b reduced the number of viable cells and increased cell cycle arrest at G2/M phase upon TMZ treatment. We found a significant increase in expression of key target genes for microRNA-21 and microRNA-10b upon using targeted versus non-targeted nanoparticles. There was also significant reduction in tumor volume when using TMZ after pre-treatment with loaded nanoparticles in human GBM cell xenografts in mice. In vivo targeted nanoparticles plus different doses of TMZ showed a significant therapeutic response even at the lowest dose of TMZ, indicating that preloading cells with antagomiR-21 and antagomiR-10b increases cellular chemosensitivity towards lower TMZ doses. Future clinical applications of this combination therapy may result in improved GBM response by using lower doses of TMZ and reducing nonspecific treatment side effects.

Cytotoxicity and Bioactivity of Calcium Silicate Cements Combined with Niobium Oxide in Different Cell Lines.

PubMed

Mestieri, Leticia Boldrin; Gomes-Cornélio, Ana Lívia; Rodrigues, Elisandra Márcia; Faria, Gisele; Guerreiro-Tanomaru, Juliane Maria; Tanomaru-Filho, Mário

2017-01-01

The aim of this study was to evaluate the cytotoxicity and bioactivity of calcium silicate-based cements combined with niobium oxide (Nb2O5) micro and nanoparticles, comparing the response in different cell lines. This evaluation used four cell lines: two primary cultures (human dental pulp cells - hDPCs and human dental follicle cells - hDFCs) and two immortalized cultures (human osteoblast-like cells - Saos-2 and mouse periodontal ligament cells - mPDL). The tested materials were: White Portland Cement (PC), mineral trioxide aggregate (MTA), white Portland cement combined with microparticles (PC/Nb2O5µ) or nanoparticles (PC/Nb2O5n) of niobium oxide (Nb2O5). Cytotoxicity was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and trypan blue exclusion assays and bioactivity by alkaline phosphatase (ALP) enzyme activity. Results were analyzed by ANOVA and Tukey test (a=0.05). PC/Nb2O5n presented similar or higher cell viability than PC/Nb2O5µ in all cell lines. Moreover, the materials presented similar or higher cell viability than MTA. Saos-2 exhibited high ALP activity, highlighting PC/Nb2O5µ material at 7 days of exposure. In conclusion, calcium silicate cements combined with micro and nanoparticles of Nb2O5 presented cytocompatibility and bioactivity, demonstrating the potential of Nb2O5 as an alternative radiopacifier agent for these cements. The different cell lines had similar response to cytotoxicity evaluation of calcium silicate cements. However, bioactivity was more accurately detected in human osteoblast-like cell line, Saos-2.

MicroRNA-29 facilitates transplantation of bone marrow-derived mesenchymal stem cells to alleviate pelvic floor dysfunction by repressing elastin.

PubMed

Jin, Minfei; Wu, Yuelin; Wang, Jun; Ye, Weiping; Wang, Lei; Yin, Peipei; Liu, Wei; Pan, Chenhao; Hua, Xiaolin

2016-11-17

Pelvic floor dysfunction (PFD) is a condition affecting many women worldwide, with symptoms including stress urinary incontinence (SUI) and pelvic organ prolapse (POP). We have previously demonstrated stable elastin-expressing bone marrow-derived mesenchymal stem cells (BMSCs) attenuated PFD in rats, and aim to further study the effect of microRNA-29a-3p regulation on elastin expression and efficacy of BMSC transplantation therapy. We inhibited endogenous microRNA-29a-3p in BMSCs and investigated its effect on elastin expression by RT-PCR and Western blot. MicroRNA-29-inhibited BMSCs were then transplanted into PFD rats, accompanied by sustained release of bFGF using formulated bFGF in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP), followed by evaluation of urodynamic tests. MicroRNA-29a-3p inhibition resulted in upregulated expression and secretion of elastin in in vitro culture of BMSCs. After co-injection with PLGA-loaded bFGF NP into the PFD rats in vivo, microRNA-29a-3p-inhibited BMSCs significantly improved the urodynamic test results. Our multidisciplinary study, combining microRNA biology, genetically engineered BMSCs, and nanoparticle technology, provides an excellent stem cell-based therapy for repairing connective tissues and treating PFD.

Micro-scale and meso-scale architectural cues cooperate and compete to direct aligned tissue formation

PubMed Central

Gilchrist, Christopher L.; Ruch, David S.; Little, Dianne; Guilak, Farshid

2014-01-01

Tissue and biomaterial microenvironments provide architectural cues that direct important cell behaviors including cell shape, alignment, migration, and resulting tissue formation. These architectural features may be presented to cells across multiple length scales, from nanometers to millimeters in size. In this study, we examined how architectural cues at two distinctly different length scales, “micro-scale” cues on the order of ~1–2 μm, and “meso-scale” cues several orders of magnitude larger (>100 μm), interact to direct aligned neo-tissue formation. Utilizing a micro-photopatterning (μPP) model system to precisely arrange cell-adhesive patterns, we examined the effects of substrate architecture at these length scales on human mesenchymal stem cell (hMSC) organization, gene expression, and fibrillar collagen deposition. Both micro- and meso-scale architectures directed cell alignment and resulting tissue organization, and when combined, meso cues could enhance or compete against micro-scale cues. As meso boundary aspect ratios were increased, meso-scale cues overrode micro-scale cues and controlled tissue alignment, with a characteristic critical width (~500 μm) similar to boundary dimensions that exist in vivo in highly aligned tissues. Meso-scale cues acted via both lateral confinement (in a cell-density-dependent manner) and by permitting end-to-end cell arrangements that yielded greater fibrillar collagen deposition. Despite large differences in fibrillar collagen content and organization between μPP architectural conditions, these changes did not correspond with changes in gene expression of key matrix or tendon-related genes. These findings highlight the complex interplay between geometric cues at multiple length scales and may have implications for tissue engineering strategies, where scaffold designs that incorporate cues at multiple length scales could improve neo-tissue organization and resulting functional outcomes. PMID:25263687

Automated 3D bioassembly of micro-tissues for biofabrication of hybrid tissue engineered constructs.

PubMed

Mekhileri, N V; Lim, K S; Brown, G C J; Mutreja, I; Schon, B S; Hooper, G J; Woodfield, T B F

2018-01-12

Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.

Morphological investigations of cells that adhered to the irregular patterned polydimethylsiloxane (PDMS) surface without reagents.

PubMed

Chung, Sung Hee; Min, Junhong

2009-07-01

Polydimethylsiloxane (PDMS) surface consisting irregular pattern was investigated to develop cell-based biochip using PDMS. PDMS surface was modified with nano- and micro-combined patterns using surface deformation technology. Hydrophobicity of nano-patterned PDMS surface was sustained. Nevertheless it has irregular patterns consisting of micro- and nano-patterns. According to atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy results by immunostaining method, human mammary epithelial cells (HMEC) adhered well on irregularly patterned surface without any reagents such as gelatin and collagen, compared to commercial culture dish. It implies PDMS material can be utilized as template for cell-based biochip without any reagents.

Single cell-based analysis of torenia petal pigments by a combination of ArF excimer laser micro sampling and nano-high performance liquid chromatography (HPLC)-mass spectrometry.

PubMed

Kajiyama, Shin'ichiro; Harada, Kazuo; Fukusaki, Eiichiro; Kobayashi, Akio

2006-12-01

The molecular constituents of the petal pigments of the Torenia plant (Torenia hybrida) were analyzed on a single-cell basis by a combination of newly developed laser-microsampling and nano-flow liquid chromatography-electro spray ionization mass spectrometry (LC-ESIMS) techniques. Our method should provide a facile method for obtaining precise metabolic profiles of each cell in a single plant tissue.

A 3D analysis of oxygen transfer in a low-cost micro-bioreactor for animal cell suspension culture.

PubMed

Yu, P; Lee, T S; Zeng, Y; Low, H T

2007-01-01

A 3D numerical model was developed to study the flow field and oxygen transport in a micro-bioreactor with a rotating magnetic bar on the bottom to mix the culture medium. The Reynolds number (Re) was kept in the range of 100-716 to ensure laminar environment for animal cell culture. The volumetric oxygen transfer coefficient (k(L)a) was determined from the oxygen concentration distribution. It was found that the effect of the cell consumption on k(L)a could be negligible. A correlation was proposed to predict the liquid-phase oxygen transfer coefficient (k(Lm)) as a function of Re. The overall oxygen transfer coefficient (k(L)) was obtained by the two-resistance model. Another correlation, within an error of 15%, was proposed to estimate the minimum oxygen concentration to avoid cell hypoxia. By combination of the correlations, the maximum cell density, which the present micro-bioreactor could support, was predicted to be in the order of 10(12) cells m(-3). The results are comparable with typical values reported for animal cell growth in mechanically stirred bioreactors.

Combined acoustic and optical trapping

PubMed Central

Thalhammer, G.; Steiger, R.; Meinschad, M.; Hill, M.; Bernet, S.; Ritsch-Marte, M.

2011-01-01

Combining several methods for contact free micro-manipulation of small particles such as cells or micro-organisms provides the advantages of each method in a single setup. Optical tweezers, which employ focused laser beams, offer very precise and selective handling of single particles. On the other hand, acoustic trapping with wavelengths of about 1 mm allows the simultaneous trapping of many, comparatively large particles. With conventional approaches it is difficult to fully employ the strengths of each method due to the different experimental requirements. Here we present the combined optical and acoustic trapping of motile micro-organisms in a microfluidic environment, utilizing optical macro-tweezers, which offer a large field of view and working distance of several millimeters and therefore match the typical range of acoustic trapping. We characterize the acoustic trapping forces with the help of optically trapped particles and present several applications of the combined optical and acoustic trapping, such as manipulation of large (75 μm) particles and active particle sorting. PMID:22025990

A monolithic integrated micro direct methanol fuel cell based on sulfo functionalized porous silicon

NASA Astrophysics Data System (ADS)

Wang, M.; Lu, Y. X.; Liu, L. T.; Wang, X. H.

2016-11-01

In this paper, we demonstrate a monolithic integrated micro direct methanol fuel cell (μDMFC) for the first time. The monolithic integrated μDMFC combines proton exchange membrane (PEM) and Pt nanocatalysts, in which PEM is achieved by the functionalized porous silicon membrane and 3D Pt nanoflowers being synthesized in situ on it as catalysts. Sulfo groups functionalized porous silicon membrane serves as a PEM and a catalyst support simultaneously. The μDMFC prototype achieves an open circuit voltage of 0.3 V, a maximum power density of 5.5 mW/cm2. The monolithic integrated μDMFC offers several desirable features such as compatibility with micro fabrication techniques, an undeformable solid PEM and the convenience of assembly.

Control of stem cell fate and function by engineering physical microenvironments

PubMed Central

Kshitiz; Park, Jinseok; Kim, Peter; Helen, Wilda; Engler, Adam J; Levchenko, Andre; Kim, Deok-Ho

2012-01-01

The phenotypic expression and function of stem cells are regulated by their integrated response to variable microenvironmental cues, including growth factors and cytokines, matrix-mediated signals, and cell-cell interactions. Recently, growing evidence suggests that matrix-mediated signals include mechanical stimuli such as strain, shear stress, substrate rigidity and topography, and these stimuli have a more profound impact on stem cell phenotypes than had previously been recognized, e.g. self-renewal and differentiation through the control of gene transcription and signaling pathways. Using a variety of cell culture models enabled by micro and nanoscale technologies, we are beginning to systematically and quantitatively investigate the integrated response of cells to combinations of relevant mechanobiological stimuli. This paper reviews recent advances in engineering physical stimuli for stem cell mechanobiology and discusses how micro- and nanoscale engineered platforms can be used to control stem cell niches environment and regulate stem cell fate and function. PMID:23077731

Rich-burn, flame-assisted fuel cell, quick-mix, lean-burn (RFQL) combustor and power generation

NASA Astrophysics Data System (ADS)

Milcarek, Ryan J.; Ahn, Jeongmin

2018-03-01

Micro-tubular flame-assisted fuel cells (mT-FFC) were recently proposed as a modified version of the direct flame fuel cell (DFFC) operating in a dual chamber configuration. In this work, a rich-burn, quick-mix, lean-burn (RQL) combustor is combined with a micro-tubular solid oxide fuel cell (mT-SOFC) stack to create a rich-burn, flame-assisted fuel cell, quick-mix, lean-burn (RFQL) combustor and power generation system. The system is tested for rapid startup and achieves peak power densities after only 35 min of testing. The mT-FFC power density and voltage are affected by changes in the fuel-lean and fuel-rich combustion equivalence ratio. Optimal mT-FFC performance favors high fuel-rich equivalence ratios and a fuel-lean combustion equivalence ratio around 0.80. The electrical efficiency increases by 150% by using an intermediate temperature cathode material and improving the insulation. The RFQL combustor and power generation system achieves rapid startup, a simplified balance of plant and may have applications for reduced NOx formation and combined heat and power.

N-Myc down regulation induced differentiation, early cell cycle exit, and apoptosis in human malignant neuroblastoma cells having wild type or mutant p53.

PubMed

Janardhanan, Rajiv; Banik, Naren L; Ray, Swapan K

2009-11-01

Neuroblastomas, which mostly occur in children, are aggressive metastatic tumors of the sympathetic nervous system. The failure of the previous therapeutic regimens to target multiple components of N-Myc pathway resulted in poor prognosis. The present study investigated the efficacy of the combination of N-(4-hydroxyphenyl) retinamide (4-HPR, 0.5 microM) and genistein (GST, 25 microM) to control the growth of human neuroblastoma cells (SH-SY5Y and SK-N-BE2) harboring divergent molecular attributes. Combination of 4-HPR and GST down regulated N-Myc, Notch-1, and Id2 to induce neuronal differentiation. Transition to neuronal phenotype was accompanied by increase in expression of e-cadherin. Induction of neuronal differentiation was associated with decreased expression of hTERT, PCNA, survivin, and fibronectin. This is the first report that combination of 4-HPR and GST mediated reactivation of multiple tumor suppressors (p53, p21, Rb, and PTEN) for early cell cycle exit (due to G1/S phase arrest) in neuroblastoma cells. Reactivation of tumor suppressor(s) repressed N-Myc driven growth factor mediated angiogenic and invasive pathways (VEGF, b-FGF, MMP-2, and MMP-9) in neuroblastoma. Repression of angiogenic factors led to the blockade of components of mitogenic pathways [phospho-Akt (Thr 308), p65 NF-kappaB, and p42/44 Erk 1/2]. Taken together, the combination of 4-HPR and GST effectively blocked survival, mitogenic, and angiogenic pathways and activated proteases for apoptosis in neuroblastoma cells. These results suggested that combination of 4-HPR and GST could be effective for controlling the growth of heterogeneous human neuroblastoma cell populations.

Nanocrystalline diamond micro-anvil grown on single crystal diamond as a generator of ultra-high pressures

DOE PAGES

Samudrala, Gopi K.; Moore, Samuel L.; Velisavljevic, Nenad; ...

2016-09-29

By combining mask-less lithography and chemical vapor deposition (CVD) techniques, a novel two-stage diamond anvil has been fabricated. A nanocrystalline diamond (NCD) micro-anvil 30 μm in diameter was grown at the center of a [100]-oriented, diamond anvil by utilizing microwave plasma CVD method. The NCD micro-anvil has a diamond grain size of 115 nm and micro-focused Raman and X-ray Photoelectron spectroscopy analysis indicate sp3-bonded diamond content of 72%. Lastly, these CVD grown NCD micro-anvils were tested in an opposed anvil configuration and the transition metals osmium and tungsten were compressed to high pressures of 264 GPa in a diamond anvilmore » cell.« less

Curcumin increases the sensitivity of Paclitaxel-resistant NSCLC cells to Paclitaxel through microRNA-30c-mediated MTA1 reduction.

PubMed

Lu, Yimin; Wang, Jun; Liu, Lei; Yu, Lequn; Zhao, Nian; Zhou, Xingju; Lu, Xudong

2017-04-01

Non-small-cell lung cancer is one of the most lethal cancers in the worldwide. Although Paclitaxel-based combinational therapies have long been used as a standard treatment in aggressive non-small-cell lung cancers, Paclitaxel resistance emerges as a major clinical problem. It has been demonstrated that Curcumin from Curcuma longa as a traditional Chinese medicine can inhibit cancer cell proliferation. However, the role of Curcumin in Paclitaxel-resistant non-small-cell lung cancer cells is not clear. In this study, we investigated the effect of Curcumin on the Paclitaxel-resistant non-small-cell lung cancer cells and found that Curcumin treatment markedly increased the sensitivity of Paclitaxel-resistant non-small-cell lung cancer cells to Paclitaxel. Mechanically, the study revealed that Curcumin could reduce the expression of metastasis-associated gene 1 (MTA1) gene through upregulation of microRNA-30c in Paclitaxel-resistant non-small-cell lung cancer cells. During the course, MTA1 reduction sensitized Paclitaxel-resistant non-small-cell lung cancer cells and enhanced the effect of Paclitaxel. Taken together, our studies indicate that Curcumin increases the sensitivity of Paclitaxel-resistant non-small-cell lung cancer cells to Paclitaxel through microRNA-30c-mediated MTA1 reduction. Curcumin might be a potential adjuvant for non-small-cell lung cancer patients during Paclitaxel treatment.

Microenvironmental Regulation of Biomacromolecular Therapies

DTIC Science & Technology

2007-06-01

of novel drug delivery systems. NATURE REVIEWS | DRUG DISCOVERY VOLUME 6 | JUNE 2007 | 455 REVIEWS © 2007 Nature Publishing Group Report...direct manner to provide cell responsiveness to protein drugs . Combined delivery of survival cytokines, including stem-cell fac- tor (SCF; also known...Figure 3 | Potential strategies to engineer cell micro environments in vivo to modulate the cellular response to protein drugs . a | Delivery of anti

Detection and distribution analysis of organosilicon compounds in wood by means of SEM-EDX and micro-CT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vetter, L. de; Cnudde, V.; Masschaele, B.

This article explores the potential of a scanning electron microscope with an energy dispersive X-ray spectrometer in combination with a new non-destructive 3D visualization technique, X-ray micro-computed tomography, as detection methods for siloxanes/silanes mixtures applied as wood preservatives. In order to have a higher contrast, bromine functional silane was added to the mixture. Scots pine and beech samples were dipped or impregnated with the mixture and subsequently scanned. Both silicon and bromine were easily detectable with both techniques. Dipped siloxanes/silanes covered the cell walls partly in beech and the lumen partly or completely in Scots pine. Impregnated siloxanes/silanes could bemore » found in the cell walls of both wood species. From the results, it can be concluded that, under the circumstances as described in the article, impregnation with a siloxane is necessary to have cell wall penetration. The combination of scanning electron microscopy, energy dispersive X-ray spectroscopy and micro-computed tomography can offer important information concerning the localization of certain products inside wood. While the last of these can monitor changes in 3D, the other two techniques can provide detailed 2D information. Both techniques are complementary and provide important extra information.« less

Improving device performance of perovskite solar cells by micro-nanoscale composite mesoporous TiO2

NASA Astrophysics Data System (ADS)

Ting, Hungkit; Zhang, Danfei; He, Yihao; Wei, Shiyuan; Li, Tieyi; Sun, Weihai; Wu, Cuncun; Chen, Zhijian; Wang, Qi; Zhang, Guoyi; Xiao, Lixin

2018-02-01

In perovskite solar cells, the morphology of the porous TiO2 electron transport layer (ETL) largely determines the quality of the perovskites. Here, we chose micro-scale TiO2 (0.2 µm) and compared it with the conventional nanoscale TiO2 (20 nm) in relation to the crystallinity of perovskites. The results show that the micro-scale TiO2 is favorable for increasing the grain size of the perovskites and enhancing the light scattering. However, the oversized TiO2 results in an uneven surface. The evenness of the perovskites can be improved by nanoscale TiO2, while the crystallinity and compactness are not as good as those of the films based on micro-scale TiO2. To combine the advantages of both micro-scale and nanoscale TiO2, by mixing 0.2 µm/20 nm TiO2 with a ratio of 1:2 as the composite ETL, the device average power conversion efficiency was increased to 11.2% from 9.9% in the case of only 20 nm TiO2.

From Extended Nanofluidics to an Autonomous Solar-Light-Driven Micro Fuel-Cell Device.

PubMed

Pihosh, Yuriy; Uemura, Jin; Turkevych, Ivan; Mawatari, Kazuma; Kazoe, Yutaka; Smirnova, Adelina; Kitamori, Takehiko

2017-07-03

Autonomous micro/nano mechanical, chemical, and biomedical sensors require persistent power sources scaled to their size. Realization of autonomous micro-power sources is a challenging task, as it requires combination of wireless energy supply, conversion, storage, and delivery to the sensor. Herein, we realized a solar-light-driven power source that consists of a micro fuel cell (μFC) and a photocatalytic micro fuel generator (μFG) integrated on a single microfluidic chip. The μFG produces hydrogen by photocatalytic water splitting under solar light. The hydrogen fuel is then consumed by the μFC to generate electricity. Importantly, the by-product water returns back to the photocatalytic μFG via recirculation loop without losses. Both devices rely on novel phenomena in extended-nano-fluidic channels that ensure ultra-fast proton transport. As a proof of concept, we demonstrate that μFG/μFC source achieves remarkable energy density of ca. 17.2 mWh cm -2 at room temperature. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced mucosal and systemic immune response with intranasal immunization of mice with HIV peptides entrapped in PLG microparticles in combination with Ulex Europaeus-I lectin as M cell target.

PubMed

Manocha, Monika; Pal, Pramod Chandra; Chitralekha, K T; Thomas, Beena Elizabeth; Tripathi, Vinita; Gupta, Siddhartha Dutta; Paranjape, Ramesh; Kulkarni, Smita; Rao, D Nageswara

2005-12-01

The predominant route of HIV infection is through the sexual transmission via M cells. Most of the peptide and protein vaccines show poor transport across the epithelial barrier and are commonly administered by parenteral route. In the present study four HIV peptides from envelope (gp 41-LZ (leucine zipper), gp 41-FD (fusion domain) and gp120-C2) and regulatory (Nef) region in poly lactic-co-glycolide (PLG) micro-particle delivery were evaluated in mice of outbred and with different genetic background to compare immune response versus MHC restriction. Out of the combinational and single routes of immunization attempted, the single route maintained the IgG, IgA and sIgA in sera and washes for longer duration as compared to combinational routes in which the response was declined. The study demonstrated that single intranasal immunization offered significantly higher immune response (p<0.05) over oral and rectal mucosal routes in terms of inducing systemic as well as mucosal response. Also, the specific activity measurement of IgA and IgG in sera and sIgA in washes were correlating to the antibody titers. However, the intramuscular route of immunization generated systemic response only. The entrapment of plant lectin UEA-1 a ligand specific for M cells in micro-particle further enhanced the immune response in all the mucosal routes. The IgG isotypes generated were of IgG1 and IgG2a/2b in sera for all the peptides. The T cell proliferation response study with and without UEA-1 lectin in micro-particles showed significantly high (p<0.05) stimulation index (SI) with intranasal immunization for all the peptides from cells collected from spleen (SP), peyer's patches (PP) and lamina propria (LP) with SI in the order LP cells>PP>or=SP. The cytokine measurement profile of IL-2, IFN-gamma and IL-6 and low levels of IL-4 in the cultural supernatants of SP, PP and LP showed mixed CD4(+) Th1 and Th2 immune response. The p24 assay showed high percent inhibition of HIV-IIIB virus with sera and washes obtained from intranasal route. Thus, overall the study highlighted the combination of UEA-1 lectin with HIV peptides in micro-particles through intranasal immunization generated systemic as well as mucosal immune response.

Multifunctional Poly(L-lactide)-Polyethylene Glycol-Grafted Graphene Quantum Dots for Intracellular MicroRNA Imaging and Combined Specific-Gene-Targeting Agents Delivery for Improved Therapeutics.

PubMed

Dong, Haifeng; Dai, Wenhao; Ju, Huangxian; Lu, Huiting; Wang, Shiyan; Xu, Liping; Zhou, Shu-Feng; Zhang, Yue; Zhang, Xueji

2015-05-27

Photoluminescent (PL) graphene quantum dots (GQDs) with large surface area and superior mechanical flexibility exhibit fascinating optical and electronic properties and possess great promising applications in biomedical engineering. Here, a multifunctional nanocomposite of poly(l-lactide) (PLA) and polyethylene glycol (PEG)-grafted GQDs (f-GQDs) was proposed for simultaneous intracellular microRNAs (miRNAs) imaging analysis and combined gene delivery for enhanced therapeutic efficiency. The functionalization of GQDs with PEG and PLA imparts the nanocomposite with super physiological stability and stable photoluminescence over a broad pH range, which is vital for cell imaging. Cell experiments demonstrate the f-GQDs excellent biocompatibility, lower cytotoxicity, and protective properties. Using the HeLa cell as a model, we found the f-GQDs effectively delivered a miRNA probe for intracellular miRNA imaging analysis and regulation. Notably, the large surface of GQDs was capable of simultaneous adsorption of agents targeting miRNA-21 and survivin, respectively. The combined conjugation of miRNA-21-targeting and survivin-targeting agents induced better inhibition of cancer cell growth and more apoptosis of cancer cells, compared with conjugation of agents targeting miRNA-21 or survivin alone. These findings highlight the promise of the highly versatile multifunctional nanocomposite in biomedical application of intracellular molecules analysis and clinical gene therapeutics.

Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng Zhiming; Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia; Wang Ping

2013-02-01

Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials:more » Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.« less

Combining heavy ion radiation and artificial microRNAs to target the homologous recombination repair gene efficiently kills human tumor cells.

PubMed

Zheng, Zhiming; Wang, Ping; Wang, Hongyan; Zhang, Xiangming; Wang, Minli; Cucinotta, Francis A; Wang, Ya

2013-02-01

Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death. Copyright © 2013 Elsevier Inc. All rights reserved.

Bio-barcode gel assay for microRNA

NASA Astrophysics Data System (ADS)

Lee, Hyojin; Park, Jeong-Eun; Nam, Jwa-Min

2014-02-01

MicroRNA has been identified as a potential biomarker because expression level of microRNA is correlated with various cancers. Its detection at low concentrations would be highly beneficial for cancer diagnosis. Here, we develop a new type of a DNA-modified gold nanoparticle-based bio-barcode assay that uses a conventional gel electrophoresis platform and potassium cyanide chemistry and show this assay can detect microRNA at aM levels without enzymatic amplification. It is also shown that single-base-mismatched microRNA can be differentiated from perfectly matched microRNA and the multiplexed detection of various combinations of microRNA sequences is possible with this approach. Finally, differently expressed microRNA levels are selectively detected from cancer cells using the bio-barcode gel assay, and the results are compared with conventional polymerase chain reaction-based results. The method and results shown herein pave the way for practical use of a conventional gel electrophoresis for detecting biomolecules of interest even at aM level without polymerase chain reaction amplification.

Micro-separation toward systems biology.

PubMed

Liu, Bi-Feng; Xu, Bo; Zhang, Guisen; Du, Wei; Luo, Qingming

2006-02-17

Current biology is experiencing transformation in logic or philosophy that forces us to reevaluate the concept of cell, tissue or entire organism as a collection of individual components. Systems biology that aims at understanding biological system at the systems level is an emerging research area, which involves interdisciplinary collaborations of life sciences, computational and mathematical sciences, systems engineering, and analytical technology, etc. For analytical chemistry, developing innovative methods to meet the requirement of systems biology represents new challenges as also opportunities and responsibility. In this review, systems biology-oriented micro-separation technologies are introduced for comprehensive profiling of genome, proteome and metabolome, characterization of biomolecules interaction and single cell analysis such as capillary electrophoresis, ultra-thin layer gel electrophoresis, micro-column liquid chromatography, and their multidimensional combinations, parallel integrations, microfabricated formats, and nano technology involvement. Future challenges and directions are also suggested.

Linking micro- and macro-evolution at the cell type level: a view from the lophotrochozoan Platynereis dumerilii.

PubMed

Simakov, Oleg; Larsson, Tomas A; Arendt, Detlev

2013-09-01

Ever since the origin of the first metazoans over 600 million years ago, cell type diversification has been driven by micro-evolutionary processes at population level, leading to macro-evolution changes above species level. In this review, we introduce the marine annelid Platynereis dumerilii, a member of the lophotrochozoan clade (a key yet most understudied superphylum of bilaterians), as a suitable model system for the simultaneous study, at cellular resolution, of macro-evolutionary processes across phyla and of micro-evolutionary processes across highly polymorphic populations collected worldwide. Recent advances in molecular and experimental techniques, easy maintenance and breeding, and the fast, synchronous and stereotypical development have facilitated the establishment of Platynereis as one of the leading model species in the eco-evo-devo field. Most importantly, Platynereis allows the combination of expression profiling, morphological and physiological characterization at the single cell level. Here, we discuss recent advances in the collection of -omics data for the lab strain and for natural populations collected world-wide that can be integrated with population-specific cellular analyses to result in a cellular atlas integrating genetic, phenotypic and ecological variation. This makes Platynereis a tractable system to begin understanding the interplay between macro- and micro-evolutionary processes and cell type diversity.

Nonlinear dynamic phase contrast microscopy for microfluidic and microbiological applications

NASA Astrophysics Data System (ADS)

Denz, C.; Holtmann, F.; Woerdemann, M.; Oevermann, M.

2008-08-01

In live sciences, the observation and analysis of moving living cells, molecular motors or motion of micro- and nano-objects is a current field of research. At the same time, microfluidic innovations are needed for biological and medical applications on a micro- and nano-scale. Conventional microscopy techniques are reaching considerable limits with respect to these issues. A promising approach for this challenge is nonlinear dynamic phase contrast microscopy. It is an alternative full field approach that allows to detect motion as well as phase changes of living unstained micro-objects in real-time, thereby being marker free, without contact and non destructive, i.e. fully biocompatible. The generality of this system allows it to be combined with several other microscope techniques such as conventional bright field or fluorescence microscopy. In this article we will present the dynamic phase contrast technique and its applications in analysis of micro organismic dynamics, micro flow velocimetry and micro-mixing analysis.

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia

PubMed Central

Silva, Ricardo Jorge; Cruz, Ana Rita; Mano, Miguel

2017-01-01

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells. PMID:28394930

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia.

PubMed

Sunkavalli, Ushasree; Aguilar, Carmen; Silva, Ricardo Jorge; Sharan, Malvika; Cruz, Ana Rita; Tawk, Caroline; Maudet, Claire; Mano, Miguel; Eulalio, Ana

2017-04-01

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.

Plasma exosome microRNAs are indicative of breast cancer.

PubMed

Hannafon, Bethany N; Trigoso, Yvonne D; Calloway, Cameron L; Zhao, Y Daniel; Lum, David H; Welm, Alana L; Zhao, Zhizhuang J; Blick, Kenneth E; Dooley, William C; Ding, W Q

2016-09-08

microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.

Tailoring of the titanium surface by preparing cardiovascular endothelial extracellular matrix layer on the hyaluronic acid micro-pattern for improving biocompatibility.

PubMed

Li, Jingan; Zhang, Kun; Wu, Juejue; Zhang, Lijuan; Yang, Ping; Tu, Qiufen; Huang, Nan

2015-04-01

It has been proved that high molecular weight hyaluronic acid (HMW-HA, 1×10(6) Da) micro-strips on titanium (Ti) surface can elongate the human vascular endothelial cell (EC) morphology, subsequently enhance endothelial extracellular matrix (ECM) deposition in our previous work. The HMW-HA micro-strips were anticipated to possess good hemocompatibility and EC compatibility simultaneously. However, the single HMW-HA micro-strips on Ti substrate showed bad hemocompatibility. To solve this problem, a method combining HA micro-pattern and EC decellularization was developed, and the endothelial extracellular matrix layer on the HA micro-pattern (ECM/HAP) showed excellent hemocompatibility and endothelial progenitor cells (EPCs) compatibility (cell number: 14.3±0.5×10(5) cells/cm2>2.2±0.7×10(5) cells/cm2 on ECM/TiOH, 7.5±1.3×10(5) cells/cm2 on TiOH, 3.4±0.9×10(5) cells/cm2 on TiOH/HAP and 3.6±1.2×10(5) cells/cm2 on Ti). We also found that the ECM/HAP coating could significantly inhibit the excessive proliferation of smooth muscle cells (SMCs) (cck-8 absorption: 0.25±0.06<1.18±0.09 A.U. on ECM/TiOH, 0.87±0.15 A.U. on TiOH and 1.55±0.11 A.U. on Ti) and the attachment of macrophages (cell number: 1.3±0.1×10(3)<9.2±1.5×10(3) cells/cm2 on ECM/TiOH, 8.8±0.3×10(3) cells/cm2 on TiOH, 7.3±0.7×10(3) cells/cm2 on TiOH/HAP and 9.6±0.9×10(3) cells/cm2 on Ti in 12 h). These data suggest that the multifunctional ECM/HAP coating can be used to build the bionic human endothelial ECM on the biomaterials surface, which might provide a potential and effective method for surface modification of cardiovascular devices. Copyright © 2015. Published by Elsevier B.V.

Cytotoxic effects and aromatase inhibition by xenobiotic endocrine disrupters alone and in combination.

PubMed

Benachour, Nora; Moslemi, Safa; Sipahutar, Herbert; Seralini, Gilles-Eric

2007-07-15

Xenobiotics may cause long-term adverse effects in humans, especially at the embryonic level, raising questions about their levels of exposure, combined effects, and crucial endpoints. We are interested in the possible interactions between xenobiotic endocrine disrupters, cellular viability and androgen metabolism. Accordingly, we tested aroclor 1254 (A1254), atrazine (AZ), o,p'-DDT, vinclozolin (VZ), p,p'-DDE, bisphenol A (BPA), chlordecone (CD), nonylphenol (NP), tributylin oxide (TBTO), and diethylstilbestrol (DES) for cellular toxicity against human embryonic 293 cells, and activity against cellular aromatase, but also on placental microsomes and on the purified equine enzyme. Cellular viability was affected in 24 h by all the xenobiotics with a threshold at 50 microM (except for TBTO and DES, 10 microM threshold), and aromatase was inhibited at non-toxic doses. In combination synergism was observed reducing the threshold values of toxicity to 4-10 microM, and aromatase activity by 50% in some cases. In placental microsomes the most active xenobiotics rapidly inhibited microsomal aromatase in a manner independent of NADPH metabolism. Prolonged exposures to low doses in cells generally amplified by 50 times aromatase inhibition. These xenobiotics may act by inhibition of the active site or by allosteric effects on the enzyme. Bioaccumulation is a feature of some xenobiotics, especially chlordecone, DDT and DDE, and low level chronic exposures can also affect cell signaling mechanisms. This new information about the mechanism of action of these xenobiotics will assist in improved molecular design with a view to providing safer compounds for use in the (human) environment.

Predicting Young’s Modulus of Glass/Ceramic Sealant for Solid Oxide Fuel Cell Considering the Combined Effects of Aging, Micro-Voids and Self-Healing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Wenning N.; Sun, Xin; Khaleel, Mohammad A.

We study the temperature dependent Young’s modulus for the glass/ceramic seal material used in Solid Oxide Fuel Cells (SOFCs). With longer heat treatment or aging time during operation, further devitrification may reduce the residual glass content in the seal material while boosting the ceramic crystalline content. In the meantime, micro-voids induced by the cooling process from the high operating temperature to room temperature can potentially degrade the mechanical properties of the glass/ceramic sealant. Upon reheating to the SOFC operating temperature, possible self-healing phenomenon may occur in the glass/ceramic sealant which can potentially restore some of its mechanical properties. A phenomenologicalmore » model is developed to model the temperature dependent Young’s modulus of glass/ceramic seal considering the combined effects of aging, micro-voids, and possible self-healing. An aging-time-dependent crystalline content model is first developed to describe the increase of the crystalline content due to the continuing devitrification under high operating temperature. A continuum damage mechanics (CDM) model is then adapted to model the effects of both cooling induced micro-voids and reheating induced self-healing. This model is applied to model the glass-ceramic G18, a candidate SOFC seal material previously developed at PNNL. Experimentally determined temperature dependent Young’s modulus is used to validate the model predictions« less

Incorporation of sodium channel blocking and free radical scavenging activities into a single drug, AM-36, results in profound inhibition of neuronal apoptosis.

PubMed

Callaway, J K; Beart, P M; Jarrott, B; Giardina, S F

2001-04-01

AM-36 is a novel neuroprotective agent incorporating both antioxidant and Na(+) channel blocking actions. In cerebral ischaemia, loss of cellular ion homeostasis due to Na(+) channel activation, together with increased reactive oxygen species (ROS) production, are thought to contribute to neuronal death. Since neuronal death in the penumbra of the ischaemic lesion is suggested to occur by apoptosis, we investigated the ability of AM-36, antioxidants and Na(+) channel antagonists to inhibit toxicity induced by the neurotoxin, veratridine in cultured cerebellar granule cells (CGC's). Veratridine (10 - 300 microM) concentration-dependently reduced cell viability of cultured CGC's. Under the experimental conditions employed, cell death induced by veratridine (100 microM) possessed the characteristics of apoptosis as assessed by morphology, TUNEL staining and DNA laddering on agarose gels. Neurotoxicity and apoptosis induced by veratridine (100 microM) were inhibited to a maximum of 50% by the antioxidants, U74500A (0.1 - 10 microM) and U83836E (0.03 - 10 microM), and to a maximum of 30% by the Na(+) channel blocker, dibucaine (0.1 - 100 microM). In contrast, AM-36 (0.01 - 10 microM) completely inhibited veratridine-induced toxicity ( IC(50) 1.7 (1.5 - 1.9) microM, 95% confidence intervals (CI) in parentheses) and concentration-dependently inhibited apoptosis. These findings suggest veratridine-induced toxicity and apoptosis are partially mediated by generation of ROS. AM-36, which combines both Na(+) channel blocking and antioxidant activity, provided superior neuroprotection compared with agents possessing only one of these actions. This bifunctional profile of activity may underlie the potent neuroprotective effects of AM-36 recently found in a stroke model in conscious rats.

MicroRNA-137 inhibits growth of glioblastoma through EGFR suppression

PubMed Central

Zhang, Zhenxing; Song, Xiaofeng; Tian, He; Miao, Ye; Feng, Xu; Li, Yang; Wang, Honglei

2017-01-01

Aberrant expression of certain microRNAs (miRNAs) has been shown to contribute to the development of Glioblastoma multiforme (GBM). However, the involvement of miR-137 in the carcinogenesis of GBM has not been reported. Here, we showed that miR-137 levels in GBM tissues were significantly lower than the paired normal brain tissue in patients’ specimens. Moreover, low miR-137 levels in GBM tissue were associated with poor prognosis. In vitro, overexpression of miR-137 decreased GBM cell growth and increased cell apoptosis, while depletion of miR-137 enhanced cell growth and decreased cell apoptosis. Combined bioinformatics analysis and dual luciferase reporter assay showed that miR-137 may target the 3’-UTR of the epidermal growth factor receptor (EGFR) to reduce its protein translation, resulting in suppression of EGFR signaling in GBM cells. Together, our data suggest that reduction in miR-137 levels in GBM tissues may increase cell growth and decrease cell apoptosis, possibly through suppression of EGFR. PMID:28386374

Manufacture of micro fluidic devices by laser welding using thermal transfer printing techniques

NASA Astrophysics Data System (ADS)

Klein, R.; Klein, K. F.; Tobisch, T.; Thoelken, D.; Belz, M.

2016-03-01

Micro-fluidic devices are widely used today in the areas of medical diagnostics and drug research, as well as for applications within the process, electronics and chemical industry. Microliters of fluids or single cell to cell interactions can be conveniently analyzed with such devices using fluorescence imaging, phase contrast microscopy or spectroscopic techniques. Typical micro-fluidic devices consist of a thermoplastic base component with chambers and channels covered by a hermetic fluid and gas tight sealed lid component. Both components are usually from the same or similar thermoplastic material. Different mechanical, adhesive or thermal joining processes can be used to assemble base component and lid. Today, laser beam welding shows the potential to become a novel manufacturing opportunity for midsize and large scale production of micro-fluidic devices resulting in excellent processing quality by localized heat input and low thermal stress to the device during processing. For laser welding, optical absorption of the resin and laser wavelength has to be matched for proper joining. This paper will focus on a new approach to prepare micro-fluidic channels in such devices using a thermal transfer printing process, where an optical absorbing layer absorbs the laser energy. Advantages of this process will be discussed in combination with laser welding of optical transparent micro-fluidic devices.

Micro Corona Ionizer as an Ozone Source for Bacterial Cell Lysis

NASA Astrophysics Data System (ADS)

Lee, Eun-Hee; Lim, Hyun Jeong; Chua, Beelee; Son, Ahjeong

2015-04-01

DNA extraction is a critical process of DNA assays including polymerase chain reaction (PCR), microarrays, molecular cloning, and DNA hybridization which has been well established and can be implemented by commercial kits. DNA extraction involves cell lysis, precipitation, and purification through the combination of physical and chemical processes. Cell lysis is essential to high DNA recovery yield which can be achieved via a variety of physical, chemical, and enzymatic methods. However, these methods were originally developed for bioassays that were labor intensive, time consuming, and vulnerable to contamination and inhibition. Here, we proposed to employ a micro corona ionizer as an ozone source to lyse bacterial cells. Ozone has been well known and used as a disinfectant which allows cell lysis and DNA extraction. Previously, we have shown that a micro corona ionizer is capable of generating a significant amount of ozone. In this study, we employed the micro corona ionizer for the bacterial cell lysis which consists of a 50 μm diameter cantilever wire as the discharge cathode and a 50 μm thick copper foil as anode. Applied voltages varied from 1900 to 2200 V with corresponding corona currents from 16 to 28 μA. The resultant ozone (concentration > 0.14 ppm) generated from the micro corona ionizer was bubbled into the sample via a miniature pump. We demonstrated the cell lysis of Pseudomonas putida as the target bacterium using the micro corona ionizer. At a flow rate of 38 ml/min and applied corona voltage of 2000 V, 98.5 ± 0.2% lysis (normalized to sonication result) was achieved after 10 min. In comparison, untreated and air-treated samples showed normalized % lysis of 11.9 ± 2.4 and 36.1 ± 1.7%, respectively. We also showed that the cell lysis efficiency could be significantly increased by increasing the flow rate and the applied corona voltage. By comparing the experimental results for continuous and pulsed treatment, we verified that the percentage of lysis is primarily determined by the total ozone treatment time.

A serum microRNA signature associated with complete remission and progression after autologous stem-cell transplantation in patients with multiple myeloma

PubMed Central

Navarro, Alfons; Díaz, Tania; Tovar, Natalia; Pedrosa, Fabiola; Tejero, Rut; Cibeira, María Teresa; Magnano, Laura; Rosiñol, Laura; Monzó, Mariano; Bladé, Joan; de Larrea, Carlos Fernández

2015-01-01

We have examined serum microRNA expression in multiple myeloma (MM) patients at diagnosis and at complete response (CR) after autologous stem-cell transplantation (ASCT), in patients with stable monoclonal gammopathy of undetermined significance, and in healthy controls. MicroRNAs were first profiled using TaqMan Human MicroRNA Arrays. Differentially expressed microRNAs were then validated by individual TaqMan MicroRNA assays and correlated with CR and progression-free survival (PFS) after ASCT. Supervised analysis identified a differentially expressed 14-microRNA signature. The differential expression of miR-16 (P = 0.028), miR-17 (P = 0.016), miR-19b (P = 0.009), miR-20a (P = 0.017) and miR-660 (P = 0.048) at diagnosis and CR was then confirmed by individual assays. In addition, high levels of miR-25 were related to the presence of oligoclonal bands (P = 0.002). Longer PFS after ASCT was observed in patients with high levels of miR-19b (6 vs. 1.8 years; P < 0.001) or miR-331 (8.6 vs. 2.9 years; P = 0.001). Low expression of both miR-19b and miR-331 in combination was a marker of shorter PFS (HR 5.3; P = 0.033). We have identified a serum microRNA signature with potential as a diagnostic and prognostic tool in MM. PMID:25593199

3D tomography of cells in micro-channels

NASA Astrophysics Data System (ADS)

Quint, S.; Christ, A. F.; Guckenberger, A.; Himbert, S.; Kaestner, L.; Gekle, S.; Wagner, C.

2017-09-01

We combine confocal imaging, microfluidics, and image analysis to record 3D-images of cells in flow. This enables us to recover the full 3D representation of several hundred living cells per minute. Whereas 3D confocal imaging has thus far been limited to steady specimens, we overcome this restriction and present a method to access the 3D shape of moving objects. The key of our principle is a tilted arrangement of the micro-channel with respect to the focal plane of the microscope. This forces cells to traverse the focal plane in an inclined manner. As a consequence, individual layers of passing cells are recorded, which can then be assembled to obtain the volumetric representation. The full 3D information allows for a detailed comparison with theoretical and numerical predictions unfeasible with, e.g., 2D imaging. Our technique is exemplified by studying flowing red blood cells in a micro-channel reflecting the conditions prevailing in the microvasculature. We observe two very different types of shapes: "croissants" and "slippers." Additionally, we perform 3D numerical simulations of our experiment to confirm the observations. Since 3D confocal imaging of cells in flow has not yet been realized, we see high potential in the field of flow cytometry where cell classification thus far mostly relies on 1D scattering and fluorescence signals.

Apoptosis and TRAF-1 cleavage in Epstein-Barr virus-positive nasopharyngeal carcinoma cells treated with doxorubicin combined with a farnesyl-transferase inhibitor.

PubMed

Vicat, Jean Michel; Ardila-Osorio, Hector; Khabir, Abdelmajid; Brezak, Marie Christine; Viossat, Isabelle; Kasprzyk, Philip; Jlidi, Rachid; Opolon, Paule; Ooka, Tadamassa; Prevost, Grégoire; Huang, Dolly P; Busson, Pierre

2003-02-01

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinomas (NPC) are much more sensitive to chemotherapy than other head and neck carcinomas. Spectacular regressions are frequently observed after induction chemotherapy. However, these favorable responses are difficult to predict and often of short duration. So far there have been only few experiments to investigate the mechanisms which underline the cytotoxic effects of anti-neoplastic drugs against NPC cells. In addition, these studies were performed almost entirely on EBV-negative cell lines therefore not truly representative of NPC cells. For the first time, we have used two EBV-positive NPC tumor lines derived from a North African (C15) and a Chinese (C666-1) patient as in vitro targets for a panel of anti-neoplastic agents. Doxorubicin, taxol and in a lesser extent cis-platinum efficiently inhibited NPC cell proliferation at clinically relevant concentrations, but all three agents failed to induce apoptosis. However, massive apoptosis of C15 cells was achieved when doxorubicin (1 microM) was combined with a farnesyl-transferase inhibitor, BIM 2001 (5 microM). Moreover, this apoptotic process was associated with a caspase-dependent early cleavage of the TNF-receptor associated factor 1 (TRAF-1) molecule, a signaling adaptor which is specifically expressed in latently EBV-infected cells. TRAF-1 cleavage might become a useful indicator of chemo-induced apoptosis in EBV-associated NPCs.

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation.

PubMed

Wang, Xiuli; Cui, Fuai; Madhu, Vedavathi; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun

2011-02-01

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and β-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

DOE PAGES

Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; ...

2014-08-20

Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155more » and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.« less

MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots

PubMed Central

Jacques, Eveline; Lewandowski, Michal; Buytaert, Jan; Fierens, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

2013-01-01

The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton. PMID:23656865

Experience curve development and cost reduction disaggregation for fuel cell markets in Japan and the US

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Max; Smith, Sarah J.; Sohn, Michael D.

Technology learning rates can be dynamic quantities as a technology moves from early development to piloting and from low volume manufacturing to high volume manufacturing. This work describes a generalizable technology analysis approach for disaggregating observed technology cost reductions and presents results of this approach for one specific case study (micro-combined heat and power fuel cell systems in Japan). We build upon earlier reports that combine discussion of fuel cell experience curves and qualitative discussion of cost components by providing greater detail on the contributing mechanisms to observed cost reductions, which were not quantified in earlier reports. Greater standardization ismore » added to the analysis approach, which can be applied to other technologies. This paper thus provides a key linkage that has been missing from earlier literature on energy-related technologies by integrating the output of earlier manufacturing cost studies with observed learning rates to quantitatively estimate the different components of cost reduction including economies of scale and cost reductions due to product performance and product design improvements. This work also provides updated fuel cell technology price versus volume trends from the California Self-Generation Incentive Program, including extensive data for solid-oxide fuel cells (SOFC) reported here for the first time. The Japanese micro-CHP market is found to have a learning rate of 18% from 2005 to 2015, while larger SOFC fuel cell systems (200 kW and above) in the California market are found to have a flat (near-zero) learning rate, and these are attributed to a combination of exogenous, market, and policy factors.« less

Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine-induced apoptosis.

PubMed

Jarmalavičiūtė, Akvilė; Tunaitis, Virginijus; Pivoraitė, Ugnė; Venalis, Algirdas; Pivoriūnas, Augustas

2015-07-01

Stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) have unique neurogenic properties that could be potentially exploited for therapeutic use. The importance of paracrine SHED signaling for neuro-regeneration has been recognized, but the exact mechanisms behind these effects are presently unknown. In the present study, we investigated the neuro-protective potential of exosomes and micro-vesicles derived from SHEDs on human dopaminergic neurons during oxidative stress-induced by 6-hydroxy-dopamine (6-OHDA). ReNcell VM human neural stem cells were differentiated into dopaminergic neurons and treated with 100 μmol/L of 6-OHDA alone or in combination with exosomes or micro-vesicles purified by ultracentrifugation from SHEDs cultivated in serum-free medium under two conditions: in standard two-dimensional culture flasks or on laminin-coated micro-carriers in a bioreactor. Real-time monitoring of apoptosis was performed with the use of time-lapse confocal microscopy and the CellEvent Caspase-3/7 green detection reagent. Exosomes but not micro-vesicles derived from SHEDs grown on the laminin-coated three-dimensional alginate micro-carriers suppressed 6-OHDA-induced apoptosis in dopaminergic neurons by approximately 80% throughout the culture period. Strikingly, no such effects were observed for the exosomes derived from SHEDs grown under standard culture conditions. Our results suggest that exosomes derived from SHEDs are considered as new potential therapeutic tool in the treatment of Parkinson's disease. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Inhibition by salmeterol and cilomilast of fluticasone-enhanced IP-10 release in airway epithelial cells.

PubMed

Reddy, P J; Aksoy, Mark O; Yang, Yi; Li, Xiu Xia; Ji, Rong; Kelsen, Steven G

2008-02-01

The CXC chemokines, IP-10/CXCL10 and IL-8/CXCL8, play a role in obstructive lung disease by attracting Th1/Tc1 lymphocytes and neutrophils, respectively. Inhaled corticosteroids (ICS) and long acting beta 2-agonists (LABA) are widely used. However, their effect(s) on the release of IP-10 and IL-8 by airway epithelial cells are poorly understood. This study examined the effects of fluticasone, salmeterol, and agents which raise intracellular cAMP (cilomilast and db-cAMP) on the expression of IP-10 and IL-8 protein and mRNA. Studies were performed in cultured human airway epithelial cells during cytokine-stimulated IP-10 and IL-8 release. Cytokine treatment (TNF-alpha, IL-1beta and IFN-gamma) increased IP-10 and IL-8 protein and mRNA levels. Fluticasone (0.1 nM to 1 microM) increased IP-10 but reduced IL-8 protein release without changing IP-10 mRNA levels assessed by real time RT-PCR. The combination of salmeterol (1 micro M) and cilomilast (1-10 mu M) reduced IP-10 but had no effect on IL-8 protein. Salmeterol alone (1 micro M) and db-cAMP alone (1 mM) antagonised the effects of fluticasone on IP-10 but not IL-8 protein. In human airway epithelial cells, inhibition by salmeterol of fluticasone-enhanced IP-10 release may be an important therapeutic effect of the LABA/ICS combination not present when the two drugs are used separately.

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D.; Sung, Derek C.; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D.; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

2016-01-01

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation. PMID:27764804

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells.

PubMed

Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D; Sung, Derek C; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

2016-11-29

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3'UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

Suppression of miR-19b enhanced the cytotoxic effects of mTOR inhibitors in human neuroblastoma cells.

PubMed

Chen, Yun; Tsai, Ya-Hui; Tseng, Bor-Jiun; Pan, Hsin-Yen; Tseng, Sheng-Hong

2016-11-01

Mammalian target of rapamycin (mTOR) inhibitors exert significant antitumor effects on several cancer cell types. In this study, we investigated the effects of mTOR inhibitors, in particular the regulation of the microRNA, in neuroblastoma cells. AZD8055 (a new mTOR inhibitor)- or rapamycin-induced cytotoxic effects on neuroblastoma cells were studied. Western blotting was used to investigate the expression of various proteins in the mTOR pathway. MicroRNA precursors and antagomirs were transfected into cells to manipulate the expression of target microRNA. AZD8055 exerted stronger cytotoxic effects than rapamycin in neuroblastoma cells (p<0.03). In addition, AZD8055 suppressed the mTOR pathway and increased the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the neuroblastoma cells. AZD8055 significantly decreased miR-19b expression (p<0.005); in contrast, rapamycin increased miR-19b expression (p<0.05). Transfection of miR-19b antagomir into the neuroblastoma cells mimicked the effects of AZD8055 treatment, whereas miR-19b overexpression reversed the effects of AZD8055. Combination of miR-19b knockdown and rapamycin treatment significantly improved the sensitivity of neuroblastoma cells to rapamycin (p<0.02). Suppression of miR-19b may enhance the cytotoxic effects of mTOR inhibitors in neuroblastoma cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Ebola virus encodes a miR-155 analog to regulate importin-α5 expression.

PubMed

Liu, Yuanwu; Sun, Jing; Zhang, Hongwen; Wang, Mingming; Gao, George Fu; Li, Xiangdong

2016-10-01

The 2014 outbreak of Ebola virus caused more than 10,000 human deaths. Current knowledge of suitable drugs, clinical diagnostic biomarkers and molecular mechanisms of Ebola virus infection is either absent or insufficient. By screening stem-loop structures from the viral genomes of four virulence species, we identified a novel, putative viral microRNA precursor that is specifically expressed by the Ebola virus. The sequence of the microRNA precursor was further confirmed by mining the existing RNA-Seq database. Two putative mature microRNAs were predicted and subsequently validated in human cell lines. Combined with this prediction of the microRNA target, we identified importin-α5, which is a key regulator of interferon signaling following Ebola virus infection, as one putative target. We speculate that this microRNA could facilitate the evasion of the host immune system by the virus. Moreover, this microRNA might be a potential clinical therapeutic target or a diagnostic biomarker for Ebola virus.

Hydrocaffeic and p-coumaric acids, natural phenolic compounds, inhibit UV-B damage in WKD human conjunctival cells in vitro and rabbit eye in vivo.

PubMed

Larrosa, Mar; Lodovici, Maura; Morbidelli, Lucia; Dolara, Piero

2008-10-01

This paper studied the effect on UV-B ocular damage of 10microM hydrocaffeic acid (HCAF) alone and as a mixture (MIX) (5 microM HCAF+5 microM p-coumaric acid). Since ocular UV-B damage is mediated by reactive oxygen species, the aim was to test if HCAF and MIX could reduce oxidation damage in human conjunctival cells (WKD) in vitro and in cornea and sclera of rabbits in vivo. After UVB irradiation (44 J/m(2)) of WKD cells, 8-oxodG levels in DNA were markedly increased and this effect was attenuated by HCAF and MIX. Rabbit eyes were treated by application of HCAF and MIX drops before UV-B exposure (79 J/m(2)). Corneal and scleral DNA oxidation damage, xanthine-oxidase (XO) activity and malondialdehyde levels (MDA) in corneal tissue and prostaglandin E(2) (PGE(2)) in the aqueous humour were reduced by HCAF alone and in combination with p-coumaric acid, showing their potential as a topical treatment against UV-B damage.

Fabrication, characterization and biomedical application of two-nozzle electrospun polycaprolactone/zein-calcium lactate composite nonwoven mat.

PubMed

Liao, Nina; Joshi, Mahesh Kumar; Tiwari, Arjun Prasad; Park, Chan-Hee; Kim, Cheol Sang

2016-07-01

The objective of the current work is to incorporate calcium lactate (CL) into polycaprolactone (PCL)/zein composite micro/nanofibrous scaffolds via electrospinning to engineer bone tissue. In this study, a composite micro/nano fibrous scaffold was fabricated using a single two-nozzle electrospinning system to combine indicative nanofibers from a blended solution of zein-CL and micro-sized fibers from a PCL solution. Incorporation of the CL into the PCL/zein fibers were shown to improve the wettability, tensile strength and biological activity of the composite mats. Moreover, the composite mats have a high efficiency to nucleate calcium phosphate from simulated body fluid (SBF) solution. An in vitro cell culture with osteoblast cells demonstrated that the electrospun composite mats possessed improved biological properties, including a better cell adhesion, spread and proliferation. This study has demonstrated that the PCL/zein-CL composite provides a simple platform to fabricate a new biomimetic scaffold for bone tissue engineering, which can recapitulate both the morphology of extracellular matrix and composition of the bone. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells

PubMed Central

Nair, Maya S; Mony, Ullas; Menon, Deepthy; Koyakutty, Manzoor; Sidharthan, Neeraj; Pavithran, Keechilat; Nair, Shantikumar V; Menon, Krishnakumar N

2015-01-01

Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-l-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34+/CD38−/CD33− phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27Kip1 leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27Kip1 in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells. PMID:26028971

Ablation of MCL1 expression by virally induced microRNA-29 reverses chemoresistance in human osteosarcomas

PubMed Central

Osaki, Shuhei; Tazawa, Hiroshi; Hasei, Joe; Yamakawa, Yasuaki; Omori, Toshinori; Sugiu, Kazuhisa; Komatsubara, Tadashi; Fujiwara, Tomohiro; Sasaki, Tsuyoshi; Kunisada, Toshiyuki; Yoshida, Aki; Urata, Yasuo; Kagawa, Shunsuke; Ozaki, Toshifumi; Fujiwara, Toshiyoshi

2016-01-01

Osteosarcoma is a rare disease diagnosed as malignant bone tumor. It is generally refractory to chemotherapy, which contributes to its poor prognosis. The reversal of chemoresistance is a major clinical challenge to improve the prognostic outcome of osteosarcoma patients. We developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301 (telomelysin) and assessed its synergistic effects with chemotherapeutic agents (cisplatin and doxorubicin) using human osteosarcoma cell lines and a xenograft tumor model. The molecular mechanism underlying the chemosensitizing effect of OBP-301 was evaluated in aspects of apoptosis induction. OBP-301 inhibits anti-apoptotic myeloid cell leukemia 1 (MCL1) expression, which in turn leads to chemosensitization in human osteosarcoma cells. The siRNA-mediated knockdown of MCL1 expression sensitized human osteosarcoma cells to common chemotherapeutic agents. We also found that upregulation of microRNA-29 targeting MCL1 via virally induced transcriptional factor E2F-1 activation was critical for the enhancement of chemotherapy-induced apoptosis in osteosarcoma cells. Telomerase-specific oncolytic adenovirus synergistically suppressed the viability of human osteosarcoma cells in combination with chemotherapeutic agents. The combination treatment also significantly inhibited tumor growth, as compared to monotherapy, in an osteosarcoma xenograft tumor model. Our data suggest that replicative virus-mediated tumor-specific MCL1 ablation may be a promising strategy to attenuate chemoresistance in osteosarcoma patients. PMID:27356624

Influence of particle geometry and PEGylation on phagocytosis of particulate carriers.

PubMed

Mathaes, Roman; Winter, Gerhard; Besheer, Ahmed; Engert, Julia

2014-04-25

Particle geometry of micro- and nanoparticles has been identified as an important design parameter to influence the interaction with cells such as macrophages. A head to head comparison of elongated, non-spherical and spherical micro- and nanoparticles with and without PEGylation was carried out to benchmark two phagocytosis inhibiting techniques. J774.A1 macrophages were incubated with fluorescently labeled PLGA micro- and nanoparticles and analyzed by confocal laser scanning microscope (CLSM) and flow cytometry (FACS). Particle uptake into macrophages was significantly reduced upon PEGylation or elongated particle geometry. A combination of both, an elongated shape and PEGylation, had the strongest phagocytosis inhibiting effect for nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

The potential of nanofibers in tissue engineering and stem cell therapy.

PubMed

Gholizadeh-Ghaleh Aziz, Shiva; Gholizadeh-Ghaleh Aziz, Sara; Akbarzadeh, Abolfazl

2016-08-01

Electrospinning is a technique in which materials in solution are shaped into continuous nano- and micro-sized fibers. Combining stem cells with biomaterial scaffolds and nanofibers affords a favorable approach for bone tissue engineering, stem cell growth and transfer, ocular surface reconstruction, and treatment of congenital corneal diseases. This review seeks to describe the current examples of the use of scaffolds in stem cell therapy. Stem cells are classified as adult or embryonic stem (ES) cells, and the advantages and drawbacks of each group are detailed. The nanofibers and scaffolds are further classified in Tables I and II , which describe specific examples from the literature. Finally, the current applications of biomaterial scaffolds containing stem cells for tissue engineering applications are presented. Overall, this review seeks to give an overview of the biomaterials available for use in combination with stem cells, and the application of nanofibers in stem cell therapy.

Transplantation of mature adipocyte-derived dedifferentiated fat cells into three-wall defects in the rat periodontium induces tissue regeneration.

PubMed

Suzuki, Daigo; Akita, Daisuke; Tsurumachi, Niina; Kano, Koichiro; Yamanaka, Katsuyuki; Kaneko, Tadashi; Kawano, Eisuke; Iguchi, Shinya; Toriumi, Taku; Arai, Yoshinori; Matsumoto, Taro; Sato, Shuichi; Honda, Masaki

2017-01-01

The transplantation of dedifferentiated fat (DFAT) cells in combination with poly(d,l-lactic-co-glycolic acid) (PLGA) scaffolds has previously been proven as an effective approach in promoting periodontal tissue regeneration in a rat fenestration defect model. The aim of this study was to assess the regenerative potential of DFAT cells in a rat model of three-wall periodontal bone defect. Three-wall bone defects were created bilaterally on the mesial side of rat maxillary first molars and were either left untreated or treated by implantation of PLGA scaffolds with DFAT cells or PLGA alone. Four weeks after surgery, the tissues were processed for micro-computed tomography (micro-CT) and histomorphometric examination. Micro-CT revealed that the PLGA/DFAT group had significantly higher rates of bone regeneration than the other groups, while histomorphometric analysis showed that the PLGA/DFAT group had significantly higher densities of collagen fiber bundles in acellular and cellular cementum than the PLGA group. Moreover, the results indicate that the placement of the PLGA scaffold prevented the downgrowth of the junctional epithelium. These findings suggest that DFAT cells contribute to tissue regeneration in three-wall periodontal defects, while PLGA provides space necessary for periodontal tissue restoration.

TU-H-CAMPUS-TeP2-03: High Sensitivity and High Resolution Fiber Based Micro-Detector for Sub-Millimeter Preclinical Dosimetry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Izaguirre, E; Pokhrel, S; Knewtson, T

2016-06-15

Purpose: Current precision of small animal and cell micro-irradiators has continuously increased during the past years. Currently, preclinical irradiators can deliver sub-millimeter fields with micrometric precision but there are no water equivalent dosimeters to determine small field profiles and dose in the orthovoltage range of energies with micrometric resolution and precision. We have developed a fiber based micro-dosimeter with the resolution and dosimetric accuracy required for radiobiological research. Methods: We constructed two prototypes of micro-dosimeters based on different compositions of fiber scintillators to study the spatial resolution and dosimetric precision of small animal and cell micro-irradiators. The first has greenmore » output and the second has blue output. The blue output dosimeter has the highest sensitivity because it matches the spectral sensitivity of silicon photomultipliers. A blue detector with 500um cross section was built and tested respect to a CC01 ion chamber, film, and the 1500um green output detector. Orthovoltage fields from 1×1mm2 to 5×5mm2 were used for detector characteristics comparison. Results: The blue fiber dosimeter shows great agreement with films and matches dose measurements with the gold-standard ion chamber for 5×5mm2 fields. The detector has the appropriate sensitivity to measure fields from 1×1mm2 to larger sizes with a 1% dosimetric accuracy. The spatial resolution is in the sub-millimeter range and the spectral matching with the photomultiplier allows reducing the sensor cross section even further than the presented prototype. These results suggest that scintillating fibers combined with silicon photomultipliers is the appropriate technology to pursue micro-dosimetry for small animals and disperse cell samples. Conclusion: The constructed detectors establish a new landmark for the resolution and sensitivity of fiber based microdetectors. The validation of the detector in our small animal and cell irradiator shows that they are appropriate for preclinical and micro single cell irradiation quality assurance and dosimetry.« less

Measurement of intracellular chloride in guinea-pig vas deferens by ion analysis, 36chloride and micro-electrodes

PubMed Central

Aickin, C. Claire; Brading, Alison F.

1982-01-01

1. Cl-sensitive micro-electrodes were used to measure the intracellular Cl activity (aCli) in smooth muscle cells of the guinea-pig vas deferens. The values obtained were compared with those of intracellular Cl (Cli) found by both ion analysis and 36Cl efflux. 2. Various combinations of filling solution for recording membrane potential (Em), and type of micro-electrode were tested. The most successful, which allowed continuous recording of aCli for several hours, was a double-barrelled electrode using the reference liquid ion exchanger (RLIE; Thomas & Cohen, 1981). However, aCli measured both by simultaneous impalements of separate cells with Cl-sensitive and conventional micro-electrodes, and by double-barrelled micro-electrodes, was about 42 mM in normal Krebs solution. This is five times higher than the value from a passive distribution. ECl was about -24 mV, more than 40 mV positive to Em. 3. On complete removal of extracellular Cl (Clo), aCli fell to an apparent level of about 3 mM. If this represents interference from other anions, the maximum error in ECl measured in normal Krebs solution is 2·5 mV. Replacement of Clo caused a rapid increase in aCli. This must be caused by an active transport of Cl- ions into the cell against their electrochemical gradient. 4. The stabilized values of aCli measured at different levels of Clo agree surprisingly well with aCli estimated from ion analysis and 36Cl efflux, assuming that the intracellular activity coefficient was the same as measured in the normal Krebs solution. The relationship of aCli to Clo was hyperbolic. 5. It is concluded that Cl-sensitive micro-electrodes accurately measure aCli in smooth muscle cells. The remarkable agreement between the direct and indirect methods of measuring Cli suggests that Cl- ions are not bound to a significant extent and that the compartment seen by the micro-electrodes is probably representative of the whole cell. PMID:7108787

Micro/Nano Fabricated Solid-State Thermoelectric Generator Devices for Integrated High Voltage Power Sources

NASA Astrophysics Data System (ADS)

Fleurial, J.-P.; Ryan, M. A.; Snyder, G. J.; Huang, C.-K.; Whitacre, J. F.; Patel, J.; Lim, J.; Borshchevsky, A.

2002-01-01

Deep space missions have a strong need for compact, high power density, reliable and long life electrical power generation and storage under extreme temperature conditions. Except for electrochemical batteries and solar cells, there are currently no available miniaturized power sources. Conventional power generators devices become inefficient in extreme environments (such as encountered in Mars, Venus or outer planet missions) and rechargeable energy storage devices can only be operated in a narrow temperature range thereby limiting mission duration. The planned development of much smaller spacecrafts incorporating a variety of micro/nanodevices and miniature vehicles will require novel, reliable power technologies. It is also expected that such micro power sources could have a wide range of terrestrial applications, in particular when the limited lifetime and environmental limitations of batteries are key factors. Advanced solid-state thermoelectric combined with radioisotope or waste heat sources and low profile energy storage devices are ideally suited for these applications. The Jet Propulsion Laboratory has been actively pursuing the development of thermoelectric micro/nanodevices that can be fabricated using a combination of electrochemical deposition and integrated circuit processing techniques. Some of the technical challenges associated with these micro/nanodevice concepts, their expected level of performance and experimental fabrication and testing results to date are presented and discussed.

Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

PubMed Central

Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

2015-01-01

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated. PMID:25523586

Micro-Spectroscopic Imaging of Lignin-Carbohydrate Complexes in Plant Cell Walls and Their Migration During Biomass Pretreatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Yining; Zhao, Shuai; Wei, Hui

2015-04-27

In lignocellulosic biomass, lignin is the second most abundant biopolymer. In plant cell walls, lignin is associated with polysaccharides to form lignin-carbohydrate complexes (LCC). LCC have been considered to be a major factor that negatively affects the process of deconstructing biomass to simple sugars by cellulosic enzymes. Here, we report a micro-spectroscopic approach that combines fluorescence lifetime imaging microscopy and Stimulated Raman Scattering microscopy to probe in situ lignin concentration and conformation at each cell wall layer. This technique does not require extensive sample preparation or any external labels. Using poplar as a feedstock, for example, we observe variation ofmore » LCC in untreated tracheid poplar cell walls. The redistribution of LCC at tracheid poplar cell wall layers is also investigated when the chemical linkages between lignin and hemicellulose are cleaved during pretreatment. Our study would provide new insights into further improvement of the biomass pretreatment process.« less

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

NASA Astrophysics Data System (ADS)

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.

Toward the human cellular microRNAome.

PubMed

McCall, Matthew N; Kim, Min-Sik; Adil, Mohammed; Patil, Arun H; Lu, Yin; Mitchell, Christopher J; Leal-Rojas, Pamela; Xu, Jinchong; Kumar, Manoj; Dawson, Valina L; Dawson, Ted M; Baras, Alexander S; Rosenberg, Avi Z; Arking, Dan E; Burns, Kathleen H; Pandey, Akhilesh; Halushka, Marc K

2017-10-01

MicroRNAs are short RNAs that serve as regulators of gene expression and are essential components of normal development as well as modulators of disease. MicroRNAs generally act cell-autonomously, and thus their localization to specific cell types is needed to guide our understanding of microRNA activity. Current tissue-level data have caused considerable confusion, and comprehensive cell-level data do not yet exist. Here, we establish the landscape of human cell-specific microRNA expression. This project evaluated 8 billion small RNA-seq reads from 46 primary cell types, 42 cancer or immortalized cell lines, and 26 tissues. It identified both specific and ubiquitous patterns of expression that strongly correlate with adjacent superenhancer activity. Analysis of unaligned RNA reads uncovered 207 unknown minor strand (passenger) microRNAs of known microRNA loci and 495 novel putative microRNA loci. Although cancer cell lines generally recapitulated the expression patterns of matched primary cells, their isomiR sequence families exhibited increased disorder, suggesting DROSHA- and DICER1-dependent microRNA processing variability. Cell-specific patterns of microRNA expression were used to de-convolute variable cellular composition of colon and adipose tissue samples, highlighting one use of these cell-specific microRNA expression data. Characterization of cellular microRNA expression across a wide variety of cell types provides a new understanding of this critical regulatory RNA species. © 2017 McCall et al.; Published by Cold Spring Harbor Laboratory Press.

Arctigenin in combination with quercetin synergistically enhances the antiproliferative effect in prostate cancer cells.

PubMed

Wang, Piwen; Phan, Tien; Gordon, David; Chung, Seyung; Henning, Susanne M; Vadgama, Jaydutt V

2015-02-01

We investigated whether a combination of two promising chemopreventive agents arctigenin (Arc) and quercetin (Q) increases the anticarcinogenic potency at lower concentrations than necessary when used individually in prostate cancer. Androgen-dependent LAPC-4 and LNCaP prostate cancer cells were treated with low doses of Arc and Q alone or in combination for 48 h. The antiproliferative activity of Arc was 10- to 20-fold stronger than Q in both cell lines. Their combination synergistically enhanced the antiproliferative effect, with a stronger effect in androgen receptor (AR) wild-type LAPC-4 cells than in AR mutated LNCaP cells. Arc demonstrated a strong ability to inhibit AR protein expression in LAPC-4 cells. The combination treatment significantly inhibited both AR and PI3K/Akt pathways compared to control. A protein array analysis revealed that the mixture targets multiple pathways particularly in LAPC-4 cells including Stat3 pathway. The mixture significantly inhibited the expression of several oncogenic microRNAs including miR-21, miR-19b, and miR-148a compared to control. The mixture also enhanced the inhibition of cell migration in both cell lines compared to individual compounds tested. The combination of Arc and Q that target similar pathways, at low physiological doses, provides a novel regimen with enhanced chemoprevention in prostate cancer. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Arctigenin in combination with quercetin synergistically enhances the anti-proliferative effect in prostate cancer cells

PubMed Central

Wang, Piwen; Phan, Tien; Gordon, David; Chung, Seyung; Henning, Susanne M.; Vadgama, Jaydutt V.

2014-01-01

Scope We investigated whether a combination of two promising chemopreventive agents arctigenin and quercetin increases the anti-carcinogenic potency at lower concentrations than necessary when used individually in prostate cancer. Methods and results Androgen-dependent LAPC-4 and LNCaP prostate cancer cells were treated with low doses of arctigenin and quercetin alone or in combination for 48h. The anti-proliferative activity of arctigenin was 10-20 fold stronger than quercetin in both cell lines. Their combination synergistically enhanced the anti-proliferative effect, with a stronger effect in androgen receptor (AR) wild-type LAPC-4 cells than in AR mutated LNCaP cells. Arctigenin demonstrated a strong ability to inhibit AR protein expression in LAPC-4 cells. The combination treatment significantly inhibited both AR and PI3K/Akt pathways compared to control. A protein array analysis revealed that the mixture targets multiple pathways particularly in LAPC-4 cells including Stat3 pathway. The mixture significantly inhibited the expression of several oncogenic microRNAs including miR-21, miR-19b, and miR-148a compared to control. The mixture also enhanced the inhibition of cell migration in both cell lines compared to individual compounds tested. Conclusion The combination of arctigenin and quercetin, that target similar pathways, at low physiological doses, provides a novel regimen with enhanced chemoprevention in prostate cancer. PMID:25380086

Combustion Characterization and Model Fuel Development for Micro-tubular Flame-assisted Fuel Cells.

PubMed

Milcarek, Ryan J; Garrett, Michael J; Baskaran, Amrish; Ahn, Jeongmin

2016-10-02

Combustion based power generation has been accomplished for many years through a number of heat engine systems. Recently, a move towards small scale power generation and micro combustion as well as development in fuel cell research has created new means of power generation that combine solid oxide fuel cells with open flames and combustion exhaust. Instead of relying upon the heat of combustion, these solid oxide fuel cell systems rely on reforming of the fuel via combustion to generate syngas for electrochemical power generation. Procedures were developed to assess the combustion by-products under a wide range of conditions. While theoretical and computational procedures have been developed for assessing fuel-rich combustion exhaust in these applications, experimental techniques have also emerged. The experimental procedures often rely upon a gas chromatograph or mass spectrometer analysis of the flame and exhaust to assess the combustion process as a fuel reformer and means of heat generation. The experimental techniques developed in these areas have been applied anew for the development of the micro-tubular flame-assisted fuel cell. The protocol discussed in this work builds on past techniques to specify a procedure for characterizing fuel-rich combustion exhaust and developing a model fuel-rich combustion exhaust for use in flame-assisted fuel cell testing. The development of the procedure and its applications and limitations are discussed.

Atypical manifestation of progressive outer retinal necrosis in AIDS patient with CD4+ T-cell counts more than 100 cells/microL on highly active antiretroviral therapy.

PubMed

Vichitvejpaisal, Pornpattana; Reeponmahar, Somporn; Tantisiriwat, Woraphot

2009-06-01

Typical progressive outer retinal necrosis (PORN) is an acute ocular infectious disease in acquired immunodeficiency syndrome (AIDS) patients with extremely low CD4+ T-cell counts. It is a form of the Varicella- zoster virus (VZV) infection. This destructive infection has an extremely rapid course that may lead to blindness in affected eyes within days or weeks. Attempts at its treatment have had limited success. We describe the case of a bilateral PORN in an AIDS patient with an initial CD4+ T-cell count >100 cells/microL that developed after initiation of highly active antiretroviral therapy (HAART). A 29-year-old Thai female initially diagnosed with human immunodeficiency virus (HIV) in 1998, presented with bilaterally decreased visual acuity after initiating HAART two months earlier. Multiple yellowish spots appeared in the deep retina without evidence of intraocular inflammation or retinal vasculitis. Her CD4+ T-cell count was 127 cells/microL. She was diagnosed as having PORN based on clinical features and positive VZV in the aqueous humor and vitreous by polymerase chain reaction (PCR). Despite combined treatment with intravenous acyclovir and intravitreous ganciclovir, the patient's visual acuity worsened with no light-perception in either eye. This case suggests that PORN should be included in the differential diagnosis of reduced visual acuity in AIDS patients initiating HAART with higher CD4+ T-cell counts. PORN may be a manifestation of the immune reconstitution syndrome.

Permeation of Therapeutic Drugs in Different Formulations across the Airway Epithelium In Vitro

PubMed Central

Meindl, Claudia; Stranzinger, Sandra; Dzidic, Neira; Salar-Behzadi, Sharareh; Mohr, Stefan; Zimmer, Andreas; Fröhlich, Eleonore

2015-01-01

Background Pulmonary drug delivery is characterized by short onset times of the effects and an increased therapeutic ratio compared to oral drug delivery. This delivery route can be used for local as well as for systemic absorption applying drugs as single substance or as a fixed dose combination. Drugs can be delivered as nebulized aerosols or as dry powders. A screening system able to mimic delivery by the different devices might help to assess the drug effect in the different formulations and to identify potential interference between drugs in fixed dose combinations. The present study evaluates manual devices used in animal studies for their suitability for cellular studies. Methods Calu-3 cells were cultured submersed and in air-liquid interface culture and characterized regarding mucus production and transepithelial electrical resistance. The influence of pore size and material of the transwell membranes and of the duration of air-liquid interface culture was assessed. Compounds were applied in solution and as aerosols generated by MicroSprayer IA-1C Aerosolizer or by DP-4 Dry Powder Insufflator using fluorescein and rhodamine 123 as model compounds. Budesonide and formoterol, singly and in combination, served as examples for drugs relevant in pulmonary delivery. Results and Conclusions Membrane material and duration of air-liquid interface culture had no marked effect on mucus production and tightness of the cell monolayer. Co-application of budesonide and formoterol, applied in solution or as aerosol, increased permeation of formoterol across cells in air-liquid interface culture. Problems with the DP-4 Dry Powder Insufflator included compound-specific delivery rates and influence on the tightness of the cell monolayer. These problems were not encountered with the MicroSprayer IA-1C Aerosolizer. The combination of Calu-3 cells and manual aerosol generation devices appears suitable to identify interactions of drugs in fixed drug combination products on permeation. PMID:26274590

Micro-Organ Device

NASA Technical Reports Server (NTRS)

Sun, Wei (Inventor); Chang, Robert C. (Inventor); Starly, Binil (Inventor); Holtorf, Heidi L. (Inventor); Leslie, Julia (Inventor); Culbertson, Christopher (Inventor); Gonda, Steve R. (Inventor)

2013-01-01

A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

Micro-organ device

NASA Technical Reports Server (NTRS)

von Gustedt-Gonda, legal representative, Iris (Inventor); Holtorf, Heidi L. (Inventor); Gonda, Steve R. (Inventor); Leslie, Julia (Inventor); Chang, Robert C. (Inventor); Sun, Wei (Inventor); Starly, Binil (Inventor); Culbertson, Christopher (Inventor)

2013-01-01

A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

PubMed

Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

The effects of nicotinic and muscarinic receptor activation on patch-clamped cells in the optic tectum of Rana pipiens.

PubMed

Yu, C-J; Debski, E A

2003-01-01

Both nicotinic and muscarinic cholinergic receptors are present in the optic tectum. To begin to understand how the activation of these receptors affects visual activity patterns, we have determined the types of physiological responses induced by their activation. Using tectal brain slices from the leopard frog, we found that application of nicotine (100 microM) evoked long-lasting responses in 60% of patch-clamped tectal cells. Thirty percent of these responses consisted of an increase in spontaneous postsynaptic currents (sPSCs) and had both a glutamatergic and GABAergic component as determined by the use of 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM) and bicuculline (25 microM), respectively. Remaining response types consisted of an inward membrane current (16%) and an increase in sPSCs combined with an inward membrane current (14%). All responses could be elicited in the presence of tetrodotoxin (0.5 microM). Muscarinic receptor-mediated responses, induced by carbachol (100 microM) application after nicotinic receptor desensitization, produced responses in 70% of tectal cells. In contrast to responses elicited by nicotine, carbachol-induced responses could be evoked multiple times without significant decrement. Responses consisted of either an outward current (57%), a decrease in sPSCs (5%) or an increase in sPSCs, with (almost 6%) or without (almost 3%) an outward current. The response elicited by carbachol was not predicted by the response of the cell to nicotine. Our results suggest that nicotinic receptors are found predominantly at presynaptic locations in the optic tectum while muscarinic receptors are most often present at postsynaptic sites. We conclude that both of these receptor types could substantially modulate visual activity by changing either the input to tectal neurons or the level of their response to that input.

Expression profiling of microRNAs in human bone tissue from postmenopausal women.

PubMed

De-Ugarte, Laura; Serra-Vinardell, Jenny; Nonell, Lara; Balcells, Susana; Arnal, Magdalena; Nogues, Xavier; Mellibovsky, Leonardo; Grinberg, Daniel; Diez-Perez, Adolfo; Garcia-Giralt, Natalia

2018-01-01

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.

Nano/micro hybrid scaffold of PCL or P3HB nanofibers combined with silk fibroin for tendon and ligament tissue engineering.

PubMed

Naghashzargar, Elham; Farè, Silvia; Catto, Valentina; Bertoldi, Serena; Semnani, Dariush; Karbasi, Saeed; Tanzi, Maria Cristina

2015-07-04

A novel biodegradable nano/micro hybrid structure was obtained by electrospinning P3HB or PCL nanofibers onto a twisted silk fibroin (SF) structure, with the aim of fabricating a suitable scaffold for tendon and ligament tissue engineering. The electrospinning (ES) processing parameters for P3HB and PCL were optimized on 2D samples, and applied to produce two different nano/micro hybrid constructs (SF/ES-PCL and SF/ES-P3HB).Morphological, chemico-physical and mechanical properties of the novel hybrid scaffolds were evaluated by SEM, ATR FT-IR, DSC, tensile and thermodynamic mechanical tests. The results demonstrated that the nanofibers were tightly wrapped around the silk filaments, and the crystallinity of the SF twisted yarns was not influenced by the presence of the electrospun polymers. The slightly higher mechanical properties of the hybrid constructs confirmed an increase of internal forces due to the interaction between nano and micro components. Cell culture tests with L929 fibroblasts, in the presence of the sample eluates or in direct contact with the hybrid structures, showed no cytotoxic effects and a good level of cytocompatibility of the nano/micro hybrid structures in term of cell viability, particularly at day 1. Cell viability onto the nano/micro hybrid structures decreased from the first to the third day of culture when compared with the control culture plastic, but appeared to be higher when compared with the uncoated SF yarns. Although additional in vitro and in vivo tests are needed, the original fabrication method here described appears promising for scaffolds suitable for tendon and ligament tissue engineering.

A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

PubMed

Jebbawi, Fadi; Fayyad-Kazan, Hussein; Merimi, Makram; Lewalle, Philippe; Verougstraete, Jean-Christophe; Leo, Oberdan; Romero, Pedro; Burny, Arsene; Badran, Bassam; Martiat, Philippe; Rouas, Redouane

2014-08-06

Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation. We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes. The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo. We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

Magnetic alginate microfibers as scaffolding elements for the fabrication of microvascular-like structures.

PubMed

Sun, Tao; Shi, Qing; Huang, Qiang; Wang, Huaping; Xiong, Xiaolu; Hu, Chengzhi; Fukuda, Toshio

2018-01-15

Traditional cell-encapsulating scaffolds may elicit adverse host responses and inhomogeneity in cellular distribution. Thus, fabrication techniques for cellular self-assembly with micro-scaffold incorporation have been used recently to generate toroidal cellular modules for the bottom-up construction of vascular-like structures. The micro-scaffolds show advantage in promoting tissue formation. However, owing to the lack of annular cell micro-scaffolds, it remains a challenge to engineer micro-scale toroidal cellular modules (micro-TCMs) to fabricate microvascular-like structures. Here, magnetic alginate microfibers (MAMs) are used as scaffolding elements, where a winding strategy enables them to be formed into micro-rings as annular cell micro-scaffolds. These micro-rings were investigated for NIH/3T3 fibroblast growth as a function of surface chemistry and MAM size. Afterwards, micro-TCMs were successfully fabricated with the formation of NIH/3T3 fibroblasts and extracellular matrix layers on the three-dimensional micro-ring surfaces. Simple non-contact magnetic assembly was used to stack the micro-TCMs along a micro-pillar, after which cell fusion rapidly connected the assembled micro-TCMs into a microvascular-like structure. Endothelial cells or drugs encapsulated in the MAMs could be included in the microvascular-like structures as in vitro cellular models for vascular tissue engineering, or as miniaturization platforms for pharmaceutical drug testing in the future. Magnetic alginate microfibers functioned as scaffolding elements for guiding cell growth in micro-scale toroidal cellular modules (micro-TCMs) and provided a magnetic functionality to the micro-TCMs for non-contact 3D assembly in external magnetic fields. By using the liquid/air interface, the non-contact spatial manipulation of the micro-TCMs in the liquid environment was performed with a cost-effective motorized electromagnetic needle. A new biofabrication paradigm of construct of microvascular-like structure. The micro-tubal-shaped structures allowed direct cell-to-cell contact that solved problems of cell-encapsulating scaffolds. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Dewetting Based Fabrication of Fibrous Micro-Scaffolds as Potential Injectable Cell Carriers

PubMed Central

Song, Hokyung; Yin, Liya; Chilian, William M.; Newby, Bi-min Zhang

2014-01-01

Although regenerative medicine utilizing tissue scaffolds has made enormous strides in recent years, many constraints still hamper their effectiveness. A limitation of many scaffolds is that they form surface patches, which are not particularly effective for some types of “wounds” that are deep within tissues, e.g., stroke, myocardial infarction. In this study, we reported the generation of fibrous micro-scaffolds feasible for delivering cells by injection into the tissue parenchyma. The micro-scaffolds (widths < 100 μm) were made by dewetting of poly (lactic-coglycolic acid) thin films containing parallel strips, and cells were seeded to form cell/polymer micro-constructs during or post the micro-scaffold fabrication process. Five types of cells including rat induced vascular progenitor cells were assessed for the formation of the micro-constructs. Critical factors in forming fibrous micro-scaffolds via dewetting of polymer thin films were found to be properties of polymers and supporting substrates, temperature, and proteins in the culture medium. Also, the ability of cells to attach to the micro-scaffolds was essential for forming cell/polymer micro-constructs. Both in vitro and in vivo assessments of injecting these micro-scaffolding constructs showed, as compared to free cells, enhanced cell retention at the injected site, which could lead to improved tissue engineering and regeneration. PMID:25579969

Pathogenicity of Peptostreptococcus micros morphotypes and Prevotella species in pure and mixed culture.

PubMed

van Dalen, P J; van Deutekom-Mulder, E C; de Graaff, J; van Steenbergen, T J

1998-02-01

Recently, an atypical rough colony morphotype of Peptostreptococcus micros, a species which is found in ulcerating infections, including periodontitis, was isolated. The virulence of morphotypes alone and in combination with Prevotella intermedia and P. nigrescens was investigated both in vivo and in vitro. All strains tested induced abscesses containing fluid pus in a mouse skin model, and lesions caused by monocultures of the rough morphotype strains of P. micros were statistically significantly larger than those induced by the smooth morphotype strains. Inocula containing both morphotypes produced similar sized abscesses compared to mono-inocula containing the same bacterial load. Both Prevotella species induced small abscesses when inoculated alone, and when Pr. nigrescens was inoculated with one of the other strains, the abscesses were not significantly different from the abscesses induced by the mono-infections of this strain. Synergy, in terms of higher numbers of colony forming units (cfu) in the mixed inocula, was found for all combinations of the rough morphotypes of P. micros and both Prevotella spp. Pus from abscesses caused by combinations of Peptostreptococcus and Prevotella spp. transmitted the infection to other mice, but no abscesses were formed in mice inoculated with pus induced by mono-inocula. These results demonstrated synergic activity between both rough and smooth P. micros strains and oral Prevotella strains. The in-vitro co-culture experiments produced no evidence of growth stimulation. The effect of P. micros strains on the immune system was investigated by testing their ability to initiate luminol-dependent chemiluminescence of polymorphonuclear leucocytes in the presence and absence of human serum. In the latter, the rough morphotype strains initiated higher counts than the smooth morphotype strains. Further work is needed to elucidate the difference in virulence between the smooth and the rough morphotype cells of P. micros and the nature of the interaction with the Prevotella spp.

Involvement of miR17 pathway in glucocorticoid-induced cell death in pediatric acute lymphoblastic leukemia.

PubMed

Harada, Masako; Pokrovskaja-Tamm, Katja; Söderhäll, Stefan; Heyman, Mats; Grander, Dan; Corcoran, Martin

2012-10-01

Analysis of the microRNA transcriptome following dexa- methasone treatment of the acute lymphocytic leukemia (ALL) cell line RS4;11 showed a global down-regulation of microRNA levels. MIR17HG was rapidly down-regulated following treatment, with chromatin immunoprecipitation (ChIP) analysis demonstrating the promoter to be a direct target of glucocorticoid (GC)-transcriptional repression and revealing the miR17-92 cluster as a prime target for dexamethasone-induced repression. The loss of miR17 family expression and concomitant increases in the miR17 target Bim occurred in an additional ALL cell line SUP-B15 but not in the dexamethasone-resistant REH. Alteration of miR17 levels through up-regulation or inhibition resulted in an decrease and increase, respectively, in Bim protein levels and dexamethasone-induced cell death. Primary ex vivo ALL cells that underwent apoptosis induced by dexamethasone also down-regulated miR17 levels. Thus, down-regulation of miR17 plays an important role in glucocorticoid-induced cell death suggesting that targeting miR17 may improve the current ALL combination therapy.

Stimuli-Responsive Nano-Carrier for Co-delivery of MiR-31 and Doxorubicin to Suppress High MtEF4 Cancer.

PubMed

Wang, Fang; Zhang, Lingyun; Bai, Xiufeng; Cao, Xintao; Jiao, Xiangyu; Huang, Yan; Li, Yansheng; Qin, Yan; Wen, Yongqiang

2018-06-13

Gene interference-based therapeutics represents a fascinating challenge and shows enormous potential for cancer treatment, in which microRNA is used to correct abnormal gene. Based on the above, we introduced microRNA-31 to bind to 3' untranslated region of mtEF4, resulting in the downregulation of its messenger RNA and protein to trigger cancer cells apoptosis through mitochondria-related pathway. To achieve better therapeutic effect, a mesoporous silica nanoparticles-based controlled nanoplatform had been developed. This system was fabricated by conjugation of microRNA-31 onto doxorubicin-loaded mesoporous silica nanoparticles with a PEI/HA coating, and drug release was triggered by acidic environment of tumors. By feat of surface functionalization and tumor-specific conjugation to nanoparticles, our drug delivery system could promote intracellular accumulation of drugs via the active transport at tumor site. More importantly, microRNA-31 not only directly targeted to mtEF4 to promote cells death, but had synergistic effects when used in combination with doxorubicin, and achieved excellent superadditive effects. As such, our research might provide new insights towards detecting high mtEF4 cancer and exploiting highly effective anticancer drugs.

Small indels induced by CRISPR/Cas9 in the 5' region of microRNA lead to its depletion and Drosha processing retardance.

PubMed

Jiang, Qian; Meng, Xing; Meng, Lingwei; Chang, Nannan; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

2014-01-01

MicroRNA knockout by genome editing technologies is promising. In order to extend the application of the technology and to investigate the function of a specific miRNA, we used CRISPR/Cas9 to deplete human miR-93 from a cluster by targeting its 5' region in HeLa cells. Various small indels were induced in the targeted region containing the Drosha processing site and seed sequences. Interestingly, we found that even a single nucleotide deletion led to complete knockout of the target miRNA with high specificity. Functional knockout was confirmed by phenotype analysis. Furthermore, de novo microRNAs were not found by RNA-seq. Nevertheless, expression of the pri-microRNAs was increased. When combined with structural analysis, the data indicated that biogenesis was impaired. Altogether, we showed that small indels in the 5' region of a microRNA result in sequence depletion as well as Drosha processing retard.

Lithographic microfabrication of biocompatible polymers for tissue engineering and lab-on-a-chip applications

NASA Astrophysics Data System (ADS)

Balciunas, Evaldas; Jonusauskas, Linas; Valuckas, Vytautas; Baltriukiene, Daiva; Bukelskiene, Virginija; Gadonas, Roaldas; Malinauskas, Mangirdas

2012-06-01

In this work, a combination of Direct Laser Writing (DLW), PoliDiMethylSiloxane (PDMS) soft lithography and UV lithography was used to create cm- scale microstructured polymer scaolds for cell culture experiments out of dierent biocompatible materials: novel hybrid organic-inorganic SZ2080, PDMS elastomer, biodegradable PEG- DA-258 and SU-8. Rabbit muscle-derived stem cells were seeded on the fabricated dierent periodicity scaolds to evaluate if the relief surface had any eect on cell proliferation. An array of microlenses was fabricated using DLW out of SZ2080 and replicated in PDMS and PEG-DA-258, showing good potential applicability of the used techniques in many other elds like micro- and nano- uidics, photonics, and MicroElectroMechanical Systems (MEMS). The synergetic employment of three dierent fabrication techniques allowed to produce desired objects with low cost, high throughput and precision as well as use materials that are dicult to process by other means (PDMS and PEG-DA-258). DLW is a relatively slow fabrication method, since the object has to be written point-by-point. By applying PDMS soft lithography, we were enabled to replicate laser-fabricated scaolds for stem cell growth and micro-optical elements for lab-on-a-chip applications with high speed, low cost and good reproducible quality.

Living nano-micro fibrous woven fabric/hydrogel composite scaffolds for heart valve engineering.

PubMed

Wu, Shaohua; Duan, Bin; Qin, Xiaohong; Butcher, Jonathan T

2017-03-15

Regeneration and repair of injured or diseased heart valves remains a clinical challenge. Tissue engineering provides a promising treatment approach to facilitate living heart valve repair and regeneration. Three-dimensional (3D) biomimetic scaffolds that possess heterogeneous and anisotropic features that approximate those of native heart valve tissue are beneficial to the successful in vitro development of tissue engineered heart valves (TEHV). Here we report the development and characterization of a novel composite scaffold consisting of nano- and micro-scale fibrous woven fabrics and 3D hydrogels by using textile techniques combined with bioactive hydrogel formation. Embedded nano-micro fibrous scaffolds within hydrogel enhanced mechanical strength and physical structural anisotropy of the composite scaffold (similar to native aortic valve leaflets) and also reduced its compaction. We determined that the composite scaffolds supported the growth of human aortic valve interstitial cells (HAVIC), balanced the remodeling of heart valve ECM against shrinkage, and maintained better physiological fibroblastic phenotype in both normal and diseased HAVIC over single materials. These fabricated composite scaffolds enable the engineering of a living heart valve graft with improved anisotropic structure and tissue biomechanics important for maintaining valve cell phenotypes. Heart valve-related disease is an important clinical problem, with over 300,000 surgical repairs performed annually. Tissue engineering offers a promising strategy for heart valve repair and regeneration. In this study, we developed and tissue engineered living nano-micro fibrous woven fabric/hydrogel composite scaffolds by using textile technique combined with bioactive hydrogel formation. The novelty of our technique is that the composite scaffolds can mimic physical structure anisotropy and the mechanical strength of natural aortic valve leaflet. Moreover, the composite scaffolds prevented the matrix shrinkage, which is major problem that causes the failure of TEHV, and better maintained physiological fibroblastic phenotype in both normal and diseased HAVIC. This work marks the first report of a combination composite scaffold using 3D hydrogel enhanced by nano-micro fibrous woven fabric, and represents a promising tissue engineering strategy to treat heart valve injury. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Inertial focusing and passive micro-mixing techniques for rare cells capturing microfluidic platform

NASA Astrophysics Data System (ADS)

Phadke, Manisha; Shaner, Sebastian; Shah, Shreyas; Rodriguez, Ygnacio; Wibowo, Denni; Whulanza, Yudan; Teriete, Peter; Allen, Jeff; Kassegne, Sam

2018-02-01

Isolation and capture of rare cells continues to be a daunting task that is still looking for an innovative and efficient method. While a variety of approaches have been suggested over the past several years, immunocapturing in a microfluidic platform carries a substantial promise as shown by recent published works. In this paper, we introduced a combination of inertial focusing and passive micro-mixing through 3D chevron-type features in a microchannel to induce chaotic mixing within antibody-coated microchannels and, ultimately, promote rare cell capture. The device introduced in this work contains curved microchannels that consist of a series of staggered chevron grooves. The curved channels enable inertial focusing while the chevron grooves allow for chaotic mixing. The microfluidics platform microfabricated through soft lithography has a polydimethylsiloxane (PDMS) foundation and was thinly coated with an alginate hydrogel derivatized with streptavidin. We submitted that our qualitative and quantitative results demonstrated the potentials in advancements in rare cell isolation through this integration of two techniques.

Business Case for a Micro-Combined Heat and Power Fuel Cell System in Commercial Applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brooks, Kriston P.; Makhmalbaf, Atefe; Anderson, David M.

2013-10-30

Combined heat and power fuel cell systems (CHP-FCSs) provide consistent electrical power and hot water with greater efficiency and lower emissions than alternative sources. These systems can be used either as baseload, grid-connected, or as off-the-grid power sources. This report presents a business case for CHP-FCSs in the range of 5 to 50 kWe. Systems in this power range are considered micro-CHP-FCS. For this particular business case, commercial applications rather than residential or industrial are targeted. To understand the benefits of implementing a micro-CHP-FCS, the characteristics that determine their competitive advantage must first be identified. Locations with high electricity pricesmore » and low natural gas prices are ideal locations for micro-CHP-FCSs. Fortunately, these high spark spread locations are generally in the northeastern area of the United States and California where government incentives are already in place to offset the current high cost of the micro-CHP-FCSs. As a result of the inherently high efficiency of a fuel cell and their ability to use the waste heat that is generated as a CHP, they have higher efficiency. This results in lower fuel costs than comparable alternative small-scale power systems (e.g., microturbines and reciprocating engines). A variety of markets should consider micro-CHP-FCSs including those that require both heat and baseload electricity throughout the year. In addition, the reliable power of micro-CHP-FCSs could be beneficial to markets where electrical outages are especially frequent or costly. Greenhouse gas emission levels from micro-CHP-FCSs are 69 percent lower, and the human health costs are 99.9 percent lower, than those attributed to conventional coal-fired power plants. As a result, FCSs can allow a company to advertise as environmentally conscious and provide a bottom-line sales advantage. As a new technology in the early stages of adoption, micro-CHP-FCSs are currently more expensive than alternative technologies. As the technology gains a foothold in its target markets and demand increases, the costs will decline in response to improved manufacturing efficiencies, similar to trends seen with other technologies. Transparency Market Research forecasts suggest that the CHP-FCS market will grow at a compound annual growth rate of greater than 27 percent over the next 5 years. These production level increases, coupled with the expected low price of natural gas, indicate the economic payback period will move to less than 5 years over the course of the next 5 years. To better understand the benefits of micro-CHP-FCSs, The U.S. Department of Energy worked with ClearEdge Power to install fifteen 5-kWe fuel cells in the commercial markets of California and Oregon. Pacific Northwest National Laboratory is evaluating these systems in terms of economics, operations, and their environmental impact in real-world applications. As expected, the economic analysis has indicated that the high capital cost of the micro-CHP-FCSs results in a longer payback period than typically is acceptable for all but early-adopter market segments. However, a payback period of less than 3 years may be expected as increased production brings system cost down, and CHP incentives are maintained or improved.« less

Preparation of composite micro/nano structure on the silicon surface by reactive ion etching: Enhanced anti-reflective and hydrophobic properties

NASA Astrophysics Data System (ADS)

Zeng, Yu; Fan, Xiaoli; Chen, Jiajia; He, Siyu; Yi, Zao; Ye, Xin; Yi, Yougen

2018-05-01

A silicon substrate with micro-pyramid structure (black silicon) is prepared by wet chemical etching and then subjected to reactive ion etching (RIE) in the mixed gas condition of SF6, CHF3 and He. We systematically study the impacts of flow rates of SF6, CHF3 and He, the etching pressure and the etching time on the surface morphology and reflectivity through various characterizations. Meanwhile, we explore and obtain the optimal combination of parameters for the preparation of composite structure that match the RIE process based on the basis of micro-pyramid silicon substrate. The composite sample prepared under the optimum parameters exhibits excellent anti-reflective performance, hydrophobic, self-cleaning and anti-corrosive properties. Based on the above characteristics, the composite micro/nano structure can be applied to solar cells, photodetectors, LEDs, outdoor devices and other important fields.

Multiscale 3D manufacturing: combining thermal extrusion printing with additive and subtractive direct laser writing

NASA Astrophysics Data System (ADS)

Malinauskas, Mangirdas; Lukoševičius, Laurynas; MackevičiÅ«tÄ--, DovilÄ--; BalčiÅ«nas, Evaldas; RekštytÄ--, Sima; Paipulas, Domas

2014-05-01

A novel approach for efficient manufacturing of three-dimensional (3D) microstructured scaffolds designed for cell studies and tissue engineering applications is presented. A thermal extrusion (fused filament fabrication) 3D printer is employed as a simple and low-cost tabletop device enabling rapid materialization of CAD models out of biocompatible and biodegradable polylactic acid (PLA). Here it was used to produce cm- scale microporous (pore size varying from 100 to 400 µm) scaffolds. The fabricated objects were further laser processed in a direct laser writing (DLW) subtractive (ablation) and additive (lithography) manners. The first approach enables precise surface modification by creating micro-craters, holes and grooves thus increasing the surface roughness. An alternative way is to immerse the 3D PLA scaffold in a monomer solution and use the same DLW setup to refine its inner structure by fabricating dots, lines or a fine mesh on top as well as inside the pores of previously produced scaffolds. The DLW technique is empowered by ultrafast lasers - it allows 3D structuring with high spatial resolution in a great variety of photosensitive materials. Structure geometry on macro- to micro- scales could be finely tuned by combining these two fabrication techniques. Such artificial 3D substrates could be used for cell growth or as biocompatible-biodegradable implants. This combination of distinct material processing techniques enables rapid fabrication of diverse functional micro- featured and integrated devices. Hopefully, the proposed approach will find numerous applications in the field of ms, microfluidics, microoptics and many others.

Modeling and control for micro and nano manipulation

NASA Astrophysics Data System (ADS)

Wejinya, Uchechukwu C.

Manipulation of micro and nano entities implies the movement of micro and nano entities from an initial position (location) to the desire position (location). This operation is not only necessary, but a required task with great precision. The tools needed for the manipulation needs to be chosen properly because the capabilities of the human hand are very restricted. Smart micro and nano manipulation are becoming of great interest in many applications including medicine and industry. In industry, high precision manipulation systems are especially needed for mass production of both micro and nano systems which consist of different component in respective scales. The transition from assembling and manipulating micro and nano entities manually to mass products with high quality is only attainable by automated assembly and manipulation systems. An example is the testing of integrated circuits which can be carried out by exchanging the manipulation tool by an electric probe. Furthermore, in medical research it is customary to pick up a single cell (human, plant, or animal), and carry it to another device which is used to further analyze the cell. Consequently, the cell of interest has to be separated from the other cells and picked up using the appropriate micro/nano tool. Hence it becomes absolutely necessary that the appropriate tool be used for specific micro or nano entity manipulation and assembly. In this research, we focus on developing micro tool for manipulating micro and nano entities in liquid environment using a micro fluidic end effector system with in-situ Polyvinylidene Fluoride (PVDF) sensing. The microfluidic end effector system consists of a DC micro-diaphragm pump and compressor, one region of flexible latex tube, a Polyvinylidene Fluoride (PVDF) sensor for in-situ measurement of micro drag force, and a micro pipette. The micro pipette of the novel microfluidic end effector system has an internal diameter (ID) smaller than 20mum used for microfluidic handling and deposition of micro and nano entities such as carbon nanotubes (CNT), DNA, and for droplet control. The novel microfluidic end effector system with force sensing can significantly improve the success rate for handling/depositing micro/nano entities in the case of carbon nanotubes between micro electrodes. The design, calibration, and experimental implementation of the novel microfluidic end effector is carried out in this research. The experimental results show the success rate for carbon nanotube(s) deposition between micro electrodes can reach close to 80%. Furthermore, carbon nanotubes are of particular interest because they are good candidates for many electronics and sensing applications. The interests in using carbon nanotubes to manufacture electronics and sensors have increased in recent years because of the increase need for making electronics smaller, and their excellent electrical and mechanical properties. These potentials can be achieved if CNTs with semiconducting and metallic band structure can be successfully deposited and separated. The use of dielectrophoresis (DEP) has been established as a course towards the efficient deposition and separation of metallic carbon nanotubes from semiconducting carbon nanotubes. For this reason, this research presents a new mathematical model for dielectrophoresis and electrorotation of carbon nanotubes. Simulation results are presented in this research to validate the developed model. The combination of both the micro robotic manipulation system and the atomic force microscopy (AFM) based nano-robotic system will provide a powerful tool for micro and nano manipulation. Additional applications of this research are endless considering the rapid development of micro and nano technologies.

Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions.

PubMed

Harz, M; Rösch, P; Peschke, K-D; Ronneberger, O; Burkhardt, H; Popp, J

2005-11-01

Microbial contamination is not only a medical problem, but also plays a large role in pharmaceutical clean room production and food processing technology. Therefore many techniques were developed to achieve differentiation and identification of microorganisms. Among these methods vibrational spectroscopic techniques (IR, Raman and SERS) are useful tools because of their rapidity and sensitivity. Recently we have shown that micro-Raman spectroscopy in combination with a support vector machine is an extremely capable approach for a fast and reliable, non-destructive online identification of single bacteria belonging to different genera. In order to simulate different environmental conditions we analyzed in this contribution different Staphylococcus strains with varying cultivation conditions in order to evaluate our method with a reliable dataset. First, micro-Raman spectra of the bulk material and single bacterial cells that were grown under the same conditions were recorded and used separately for a distinct chemotaxonomic classification of the strains. Furthermore Raman spectra were recorded from single bacterial cells that were cultured under various conditions to study the influence of cultivation on the discrimination ability. This dataset was analyzed both with a hierarchical cluster analysis (HCA) and a support vector machine (SVM).

Ca(2+)-stores mobilization by diadenosine tetraphosphate, Ap4A, through a putative P2Y purinoceptor in adrenal chromaffin cells.

PubMed Central

Castro, E.; Pintor, J.; Miras-Portugal, M. T.

1992-01-01

1. Diadenosine tetraphosphate (Ap4A) evoked a concentration-dependent increase in cytosolic [Ca2+] in resting chromaffin cells. The EC50 value for this action was 28.2 +/- 6.6 microM. This effect was also produced by diadenosine pentaphosphate (Ap5A) with an EC50 of 50 +/- 7 microM. 2. In contrast with this effect, pretreatment with Ap4A or Ap5A induced a 30% reduction in Ca2+ entry following 10 microM dimethylphenylpiperazinium. 3. The elevation in cytosolic [Ca2+] induced by Ap4A was persistent in approximately 100 nM external [Ca2+] and was sensitive to depletion of internal Ca2+ stores by a bradykinin prepulse or whole cell depletion in Ca2+. 4. The effect of Ap4A was mimicked and desensitized by the agonist adenosine 5'-O-(2-thiodiphosphate), and blocked by the P2Y-receptor antagonist, cibachrome blue. The P2X-receptor agonist alpha,beta-methylene adenosine 5'-triphosphate was inactive both by itself or in combination with Ap4A. This is compatible with a P2Y-purinoceptor-mediated action. PMID:1393282

Ca(2+)-stores mobilization by diadenosine tetraphosphate, Ap4A, through a putative P2Y purinoceptor in adrenal chromaffin cells.

PubMed

Castro, E; Pintor, J; Miras-Portugal, M T

1992-08-01

1. Diadenosine tetraphosphate (Ap4A) evoked a concentration-dependent increase in cytosolic [Ca2+] in resting chromaffin cells. The EC50 value for this action was 28.2 +/- 6.6 microM. This effect was also produced by diadenosine pentaphosphate (Ap5A) with an EC50 of 50 +/- 7 microM. 2. In contrast with this effect, pretreatment with Ap4A or Ap5A induced a 30% reduction in Ca2+ entry following 10 microM dimethylphenylpiperazinium. 3. The elevation in cytosolic [Ca2+] induced by Ap4A was persistent in approximately 100 nM external [Ca2+] and was sensitive to depletion of internal Ca2+ stores by a bradykinin prepulse or whole cell depletion in Ca2+. 4. The effect of Ap4A was mimicked and desensitized by the agonist adenosine 5'-O-(2-thiodiphosphate), and blocked by the P2Y-receptor antagonist, cibachrome blue. The P2X-receptor agonist alpha,beta-methylene adenosine 5'-triphosphate was inactive both by itself or in combination with Ap4A. This is compatible with a P2Y-purinoceptor-mediated action.

Dewetting based fabrication of fibrous micro-scaffolds as potential injectable cell carriers.

PubMed

Song, Hokyung; Yin, Liya; Chilian, William M; Zhang Newby, Bi-Min

2015-03-01

Although regenerative medicine utilizing tissue scaffolds has made enormous strides in recent years, many constraints still hamper their effectiveness. A limitation of many scaffolds is that they form surface patches, which are not particularly effective for some types of "wounds" that are deep within tissues, e.g., stroke and myocardial infarction. In this study, we reported the generation of fibrous micro-scaffolds feasible for delivering cells by injection into the tissue parenchyma. The micro-scaffolds (widths<100μm) were made by dewetting of poly(lactic-co-glycolic acid) thin films containing parallel strips, and cells were seeded to form cell/polymer micro-constructs during or post the micro-scaffold fabrication process. Five types of cells including rat induced vascular progenitor cells were assessed for the formation of the micro-constructs. Critical factors in forming fibrous micro-scaffolds via dewetting of polymer thin films were found to be properties of polymers and supporting substrates, temperature, and proteins in the culture medium. Also, the ability of cells to attach to the micro-scaffolds was essential in forming cell/polymer micro-constructs. Both in vitro and in vivo assessments of injecting these micro-scaffolding constructs showed, as compared to free cells, enhanced cell retention at the injected site, which could lead to improved tissue engineering and regeneration. Copyright © 2014 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirsch, David G., E-mail: david.kirsch@duke.ed; Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA; Departments of Radiation Oncology and Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC

Purpose: To image a genetically engineered mouse model of non-small-cell lung cancer with micro-computed tomography (micro-CT) to measure tumor response to radiation therapy. Methods and Materials: The Cre-loxP system was used to generate primary lung cancers in mice with mutation in K-ras alone or in combination with p53 mutation. Mice were serially imaged by micro-CT, and tumor volumes were determined. A comparison of tumor volume by micro-CT and tumor histology was performed. Tumor response to radiation therapy (15.5 Gy) was assessed with micro-CT. Results: The tumor volume measured with free-breathing micro-CT scans was greater than the volume calculated by histology.more » Nevertheless, this imaging approach demonstrated that lung cancers with mutant p53 grew more rapidly than lung tumors with wild-type p53 and also showed that radiation therapy increased the doubling time of p53 mutant lung cancers fivefold. Conclusions: Micro-CT is an effective tool to noninvasively measure the growth of primary lung cancers in genetically engineered mice and assess tumor response to radiation therapy. This imaging approach will be useful to study the radiation biology of lung cancer.« less

MicroRNA-375/SEC23A as biomarkers of the in vitro efficacy of vandetanib.

PubMed

Lassalle, Sandra; Zangari, Joséphine; Popa, Alexandra; Ilie, Marius; Hofman, Véronique; Long, Elodie; Patey, Martine; Tissier, Frédérique; Belléannée, Geneviève; Trouette, Hélène; Catargi, Bogdan; Peyrottes, Isabelle; Sadoul, Jean-Louis; Bordone, Olivier; Bonnetaud, Christelle; Butori, Catherine; Bozec, Alexandre; Guevara, Nicolas; Santini, José; Hénaoui, Imène Sarah; Lemaire, Géraldine; Blanck, Olivier; Vielh, Philippe; Barbry, Pascal; Mari, Bernard; Brest, Patrick; Hofman, Paul

2016-05-24

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib.Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.

Numerical characterization of micro-cell UO2sbnd Mo pellet for enhanced thermal performance

NASA Astrophysics Data System (ADS)

Lee, Heung Soo; Kim, Dong-Joo; Kim, Sun Woo; Yang, Jae Ho; Koo, Yang-Hyun; Kim, Dong Rip

2016-08-01

Metallic micro-cell UO2 pellet with high thermal conductivity has received attention as a promising accident-tolerant fuel. Although experimental demonstrations have been successful, studies on the potency of current metallic micro-cell UO2 fuels for further enhancement of thermal performance are lacking. Here, we numerically investigated the thermal conductivities of micro-cell UO2sbnd Mo pellets in terms of the amount of Mo content, the unit cell size, and the aspect ratio of the micro-cells. The results showed good agreement with experimental measurements, and more importantly, indicated the importance of optimizing the unit cell geometries of the micro-cell pellets for greater increases in thermal conductivity. Consequently, the micro-cell UO2sbnd Mo pellets (5 vol% Mo) with modified geometries increased the thermal conductivity of the current UO2 pellets by about 2.5 times, and lowered the temperature gradient within the pellets by 62.9% under a linear heat generation rate of 200 W/cm.

Low dose combinations of 2-methoxyestradiol and docetaxel block prostate cancer cells in mitosis and increase apoptosis.

PubMed

Reiner, Teresita; de las Pozas, Alicia; Gomez, Lourdes A; Perez-Stable, Carlos

2009-04-08

Clinical trials have shown that chemotherapy with docetaxel (Doc) combined with prednisone can improve survival of patients with androgen-independent prostate cancer (AI-PC). It is likely that the combination of Doc with other novel agents would also improve the survival of AI-PC patients. We investigated whether the combination of Doc and 2-methoxyestradiol (2ME2), an endogenous metabolite of estradiol promising for cancer therapy, can increase apoptotic cell death in prostate cancer cells. Low concentration 2ME2 (0.5-1 microM)+Doc (0.05-0.1 nM) combinations inhibit cell growth, increase G2/M cell cycle arrest, and increase apoptosis more effectively than the single concentrations in a variety of human AI-PC cells. Effects on apoptosis were associated with an increase in p53 protein and a decrease in cyclin A-dependent kinase activity. We then investigated whether the combination of 2ME2+Doc can increase apoptotic cell death and inhibit the growth of prostate tumors in the FG/Tag transgenic mouse model of AI-PC. Doses of 2ME2 and Doc that increase mitotic cell cycle arrest result in an increase in apoptosis and lower primary prostate tumor weights in FG/Tag mice. High dose 2ME2+Doc combinations did not increase G2/M cell cycle arrest or apoptosis in AI-PC cell lines and in the FG/Tag mice more than the single drugs. Overall, our data indicate that low dose 2ME2+Doc combinations may provide a treatment strategy that can improve therapeutic efficacy against AI-PC while reducing toxicity often seen in patients treated with Doc.

MicroRNA-93 inhibits tumor growth and early relapse of human colorectal cancer by affecting genes involved in the cell cycle.

PubMed

Yang, I-Ping; Tsai, Hsiang-Lin; Hou, Ming-Feng; Chen, Ku-Chung; Tsai, Pei-Chien; Huang, Szu-Wei; Chou, Wen-Wen; Wang, Jaw-Yuan; Juo, Suh-Hang Hank

2012-08-01

Colorectal cancer (CRC) is associated with high recurrence and mortality. Because deregulation of microRNAs is associated with CRC development and recurrence, the expression levels of microRNAs can be a simple and reliable biomarker to detect postoperative early relapse, thereby helping physicians to treat high-risk patients more efficiently. We used microRNA arrays and observed that microRNA-93 had substantially different expression levels in early (recurrence within 12 months after surgery) and non-early relapse CRC patients. The replication study, which included 35 early relapse and 42 non-early relapse subjects, further confirmed overexpression of microRNA-93 in non-early relapse samples. The in vitro and in vivo effects of microRNA-93 were investigated by examining cell proliferation, migration and invasion, as well as cell cycles, target-gene expression and xenograft in null mice. Cellular studies showed that the overexpression of microRNA-93 inhibited colon cancer cell proliferation and migration but not invasion. The cell cycle studies also revealed that microRNA-93 caused an accumulation of the G2 population. However, microRNA-93 could not induce cell apoptosis or necrosis. Functional studies showed that microRNA-93 could suppress CCNB1 protein expression leading to cell cycle arrest in the G2 phase. Moreover, microRNA-93 repressed expression of ERBB2, p21 and VEGF, all of which are involved in cell proliferation. MicroRNA-93 also suppressed tumor growth in null mice. This study showed that microRNA-93 can inhibit tumorigenesis and reduce the recurrence of CRC; these findings may have potential clinical applications for predicting the recurrence of CRC.

Hierarchical structures consisting of SiO2 nanorods and p-GaN microdomes for efficiently harvesting solar energy for InGaN quantum well photovoltaic cells.

PubMed

Ho, Cheng-Han; Lien, Der-Hsien; Chang, Hung-Chih; Lin, Chin-An; Kang, Chen-Fang; Hsing, Meng-Kai; Lai, Kun-Yu; He, Jr-Hau

2012-12-07

We experimentally and theoretically demonstrated the hierarchical structure of SiO(2) nanorod arrays/p-GaN microdomes as a light harvesting scheme for InGaN-based multiple quantum well solar cells. The combination of nano- and micro-structures leads to increased internal multiple reflection and provides an intermediate refractive index between air and GaN. Cells with the hierarchical structure exhibit improved short-circuit current densities and fill factors, rendering a 1.47 fold efficiency enhancement as compared to planar cells.

Breakthrough: micro-electronic photovoltaics

ScienceCinema

Okandan, Murat; Gupta, Vipin

2018-01-16

Sandia developed tiny glitter-sized photovoltaic (PV) cells that could revolutionize solar energy collection. The crystalline silicon micro-PV cells will be cheaper and have greater efficiencies than current PV collectors. Micro-PV cells require relatively little material to form well-controlled, highly efficient devices. Cell fabrication uses common microelectric and micro-electromechanical systems (MEMS) techniques.

Nanowall formation by maskless wet-etching on a femtosecond laser irradiated silicon surface

NASA Astrophysics Data System (ADS)

Lee, Siwoo; Jo, Kukhyun; Keum, Hee-sung; Chae, Sangmin; Kim, Yonghyeon; Choi, Jiyeon; Lee, Hyun Hwi; Kim, Hyo Jung

2018-04-01

We found that micro-cells surrounded by nanowalls can be formed by a maskless wet-etching process on Si (100) surfaces possessing Laser Induced Periodic Surface Structure (LIPSS) by femtosecond laser irradiation. The LIPSS process could produce periodic one-dimensional micron scale ripples on a Si surface, which could be developed into micro-cells by a subsequent etching process. The solution etching conditions strongly affected both the micro-cell and nanowall shapes such as the height and the thickness of nanowalls. The tetramethylammonium hydroxide solution created thin nanowalls and the resulting micro-cells with a well-flattened bottom while the KOH solution formed thick walls and incomplete micro-cells. The bottoms of micro-cells surrounded by the nanowalls were considerably flat with a 3.10 nm surface roughness. A pentacene layer was deposited on the micro-cells of a Si surface to evaluate the film properties by grazing incidence wide angle x-ray scattering measurements. The pentacene film on the micro-cell Si surface showed a strong film phase, which was comparable to the film phase grown on the atomically flat Si surface.

Delivery of miR-200c Mimic with Poly(amido amine) CXCR4 Antagonists for Combined Inhibition of Cholangiocarcinoma Cell Invasiveness.

PubMed

Xie, Ying; Wehrkamp, Cody J; Li, Jing; Wang, Yan; Wang, Yazhe; Mott, Justin L; Oupický, David

2016-03-07

Cholangiocarcinoma is the second most common primary liver malignancy with extremely poor prognosis due to early invasion and widespread metastasis. The invasion and metastasis are regulated by multiple factors including CXCR4 chemokine receptor and multiple microRNAs. The goal of this study was to test the hypothesis that inhibition of CXCR4 combined with the action of miR-200c mimic will cooperatively enhance the inhibition of the invasion of human cholangiocarcinoma cells. The results show that CXCR4-inhibition polycation PCX can effectively deliver miR-200c mimic and that the combination treatment consisting of PCX and miR-200c results in cooperative antimigration activity, most likely by coupling the CXCR4 axis blockade with epithelial-to-mesenchymal transition inhibition in the cholangiocarcinoma cells. The ability of the combined PCX/miR-200c treatment to obstruct two migratory pathways represents a promising antimetastatic strategy in cholangiocarcinoma.

Suppression of ErbB-2 in androgen-independent human prostate cancer cells enhances cytotoxic effect by gemcitabine in an androgen-reduced environment.

PubMed

Zhang, Li; Davis, Jeffrey S; Zelivianski, Stanislav; Lin, Fen-Fen; Schutte, Rachel; Davis, Thomas L; Hauke, Ralph; Batra, Surinder K; Lin, Ming-Fong

2009-11-18

We examined the efficacy of combination treatments utilizing cytotoxic drugs plus inhibitors to members of the ErbB-ERK signal pathway in human prostate cancer (PCa) LNCaP C-81 cells. Under an androgen-reduced condition, 50nM gemcitabine caused about 40% growth suppression on C-81 cells. Simultaneous treatment of gemcitabine plus 10microM AG825 produced 60% suppression (p<0.03); while, 85% growth inhibition (p<0.02) was seen if AG825 was added to gemcitabine-treated cells after a 24h-interval. Our data thus showed that in androgen-reduced conditions, inhibition of ErbB-2 increases the cytotoxic efficacy of gemcitabine in PCa cells. This finding has significant implications in the choice of drugs for combination therapy as well as the order of administration for treating cancer patients.

Targeted microbubbles with ultrasound irradiation and PD-1 inhibitor to increase antitumor activity in B-cell lymphoma.

PubMed

Zheng, Shiya; Song, Dan; Jin, Xiaoxiao; Zhang, Haijun; Aldarouish, Mohanad; Chen, Yan; Wang, Cailian

2018-02-01

Severe cardiac toxicity of doxorubicin and an immunosuppressive tumor micro-environment become main obstacles for the effective treatment of B-cell lymphoma. In this research, rituximab-conjugated and doxorubicin-loaded microbubbles (RDMs) were designed for exploring a combination approach of targeted microbubbles with ultrasound (US) irradiation and PD-1 inhibitor to overcome obstacles mentioned above. In vivo studies were performed on SU-DHL-4 cell-grafted mice and ex vivo studies were performed on CD20 + human SU-DHL-4 cells and human T cells. A greater therapeutic effect and higher expression of PD-L1 protein expression were obtained with RDMs with US irradiation in vivo. A significant inhibitory effect on SU-DHL-4 B-cell lymphoma cells was observed after treated by RDMs with US irradiation and PD-1 inhibitor ex vivo. Combination of RDMs with US irradiation and PD-1 inhibitor could be a promising therapeutic strategy for B-cell lymphoma.

Fabrication of micro-cell UO2-Mo pellet with enhanced thermal conductivity

NASA Astrophysics Data System (ADS)

Kim, Dong-Joo; Rhee, Young Woo; Kim, Jong Hun; Kim, Keon Sik; Oh, Jang Soo; Yang, Jae Ho; Koo, Yang-Hyun; Song, Kun-Woo

2015-07-01

As one of accident tolerant fuel pellets which should have features of good thermal conductivity and high fission product retention, a micro-cell UO2-Mo pellet has been studied in the aspect of fabrication and thermal property. It was intended to develop the compatible process with conventional UO2 pellet fabrication process. The effects of processing parameters such as the size and density of UO2 granule and the size of Mo powder have been studied to produce sound and dense pellet with completely connected uniform Mo cell-walls. The micro-cell UO2-Mo pellet consists of many Mo micro-cells and UO2 in them. The thermal conductivity of the micro-cell UO2-Mo pellet was measured and compared to those of the UO2 pellet and the UO2-Mo pellet with dispersed form of Mo particles. The thermal conductivity of the micro-cell UO2-Mo pellet was much enhanced and was found to be influenced by the Mo volumetric fraction and pellet integrity. A continuous Mo micro-cell works as a heat conducting channel in the pellet, greatly enhancing the thermal conductivity of the micro cell UO2-Mo pellet.

Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.

PubMed

Takikawa, Tetsuya; Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Kogure, Takayuki; Shimosegawa, Tooru

2017-01-01

Pancreatic stellate cells (PSCs) interact with pancreatic cancer cells in the tumor microenvironment. Cell constituents including microRNAs may be exported from cells within membranous nanovesicles termed exosomes. Exosomes might play a pivotal role in intercellular communication. This study aimed to clarify the microRNA signature of PSC-derived exosomes and their effects on pancreatic cancer cells. Exosomes were prepared from the conditioned medium of immortalized human PSCs. MicroRNAs were prepared from the exosomes and their source PSCs, and the microRNA expression profiles were compared by microarray. The effects of PSC-derived exosomes on proliferation, migration, and the mRNA expression profiles were examined in pancreatic cancer cells. Pancreatic stellate cell-derived exosomes contained a variety of microRNAs including miR-21-5p. Several microRNAs such as miR-451a were enriched in exosomes compared to their source PSCs. Pancreatic stellate cell-derived exosomes stimulated the proliferation, migration and expression of mRNAs for chemokine (C - X - C motif) ligands 1 and 2 in pancreatic cancer cells. The stimulation of proliferation, migration, and chemokine gene expression by the conditioned medium of PSCs was suppressed by GW4869, an exosome inhibitor. We clarified the microRNA expression profile in PSC-derived exosomes. Pancreatic stellate cell-derived exosomes might play a role in the interactions between PSCs and pancreatic cancer cells.

Ultrahigh resolution optical coherence elastography combined with a rigid micro-endoscope (Conference Presentation)

NASA Astrophysics Data System (ADS)

Fang, Qi; Curatolo, Andrea; Wijesinghe, Philip; Hamzah, Juliana; Ganss, Ruth; Noble, Peter B.; Karnowski, Karol; Sampson, David D.; Kim, Jun Ki; Lee, Wei M.; Kennedy, Brendan F.

2017-02-01

The mechanical forces that living cells experience represent an important framework in the determination of a range of intricate cellular functions and processes. Current insight into cell mechanics is typically provided by in vitro measurement systems; for example, atomic force microscopy (AFM) measurements are performed on cells in culture or, at best, on freshly excised tissue. Optical techniques, such as Brillouin microscopy and optical elastography, have been used for ex vivo and in situ imaging, recently achieving cellular-scale resolution. The utility of these techniques in cell mechanics lies in quick, three-dimensional and label-free mechanical imaging. Translation of these techniques toward minimally invasive in vivo imaging would provide unprecedented capabilities in tissue characterization. Here, we take the first steps along this path by incorporating a gradient-index micro-endoscope into an ultrahigh resolution optical elastography system. Using this endoscope, a lateral resolution of 2 µm is preserved over an extended depth-of-field of 80 µm, achieved by Bessel beam illumination. We demonstrate this combined system by imaging stiffness of a silicone phantom containing stiff inclusions and a freshly excised murine liver tissue. Additionally, we test this system on murine ribs in situ. We show that our approach can provide high quality extended depth-of-field images through an endoscope and has the potential to measure cell mechanics deep in tissue. Eventually, we believe this tool will be capable of studying biological processes and disease progression in vivo.

Treatment with docetaxel in combination with Aneustat leads to potent inhibition of metastasis in a patient-derived xenograft model of advanced prostate cancer

PubMed Central

Qu, Sifeng; Ci, Xinpei; Xue, Hui; Dong, Xin; Hao, Jun; Lin, Dong; Clermont, Pier-Luc; Wu, Rebecca; Collins, Colin C; Gout, Peter W; Wang, Yuzhuo

2018-01-01

Background: Docetaxel used for first-line treatment of advanced prostate cancer (PCa) is only marginally effective. We previously showed, using the LTL-313H subrenal capsule patient-derived metastatic PCa xenograft model, that docetaxel combined with Aneustat (OMN54), a multivalent plant-derived therapeutic, led to marked synergistic tumour growth inhibition. Here, we investigated the effect of docetaxel+Aneustat on metastasis. Methods: C4-2 cells were incubated with docetaxel, Aneustat and docetaxel+Aneustat to assess effects on cell migration. The LTL-313H model, similarly treated, was analysed for effects on lung micro-metastasis and kidney invasion. The LTL-313H gene expression profile was compared with profiles of PCa patients (obtained from Oncomine) and subjected to IPA to determine involvement of cancer driver genes. Results: Docetaxel+Aneustat markedly inhibited C4-2 cell migration and LTL-313H lung micro-metastasis/kidney invasion. Oncomine analysis indicated that treatment with docetaxel+Aneustat was associated with improved patient outcome. The drug combination markedly downregulated expression of cancer driver genes such as FOXM1 (and FOXM1-target genes). FOXM1 overexpression reduced the anti-metastatic activity of docetaxel+Aneustat. Conclusions: Docetaxel+Aneustat can inhibit PCa tissue invasion and metastasis. This activity appears to be based on reduced expression of cancer driver genes such as FOXM1. Use of docetaxel+Aneustat may provide a new, more effective regimen for therapy of metastatic PCa. PMID:29381682

Priming effect of platelet activating factor on leukotriene C4 from stimulated eosinophils of asthmatic patients.

PubMed Central

Shindo, K.; Koide, K.; Hirai, Y.; Sumitomo, M.; Fukumura, M.

1996-01-01

BACKGROUND: Eosinophils from asthmatic patients are known to release greater amounts of leukotrienes than normal eosinophils when stimulated by the calcium ionophore A23187. The effect of platelet activating factor (PAF) in priming eosinophils was investigated. METHODS: Eosinophils were obtained from 18 asthmatic patients and 18 healthy donors. Cells separated by the Percoll gradients were incubated with PAF (C-18) for 30 minutes and then stimulated with the calcium ionophore A23187 (2.5 microM) for 15 minutes. The amount of leukotriene C4 (LTC4) in supernatants was measured using a combination of high pressure liquid chromatography and radioimmunoassay. RESULTS: The mean (SD) amount of LTC4 released by eosinophils from asthmatic patients upon stimulation with the calcium ionophore A23187 alone was 27.9 (9.9) ng/10(6) cells (n = 6). The amount of LTC4 released following stimulation with the calcium ionophore A23187 after pretreatment with PAF (1, 5, and 10 microM) was 57.2 (8.9), 75.1 (14.3), and 52.6 (10.7) ng/10(6) cells (n = 6), respectively. Trace amounts of LTC4 (0.9 (0.02) ng/10(6) cells, n = 6) were detected in the supernatant of the cells after stimulation by PAF alone (5 microM). The amount of LTC4 released upon stimulation by calcium ionophore A23187 alone in eosinophils from healthy donors was 10.3 (3.7) ng/10(6) cells (n = 4). The amounts of LTC4 released upon stimulation with calcium ionophore A23187 after pretreatment with PAF at concentrations of 1, 5, and 10 microM were 11.9 (3.5), 17.8 (5.6), and 12.7 (5.1) ng/10(6) cells (n = 4), respectively. Trace amounts of LTC4 (0.6 (0.02) ng/10(6) cells, n = 4) were detected in the supernatant of the cells upon stimulation with PAF alone (5 microM). The amounts of LTC4 released upon stimulation with calcium ionophore A23187 after pretreatment with lyso-PAF at concentrations of 1, 5, and 10 microM (n = 4 or 6) were 30.8 (5.2), 22.9 (5.1), and 27.3 (4.3) ng/10(6) cells (n = 6) from the eosinophils of asthmatic patients and 13.7 (3.3), 15.2 (4.9), and 14.7 (3.8) ng/10(6) cells (n = 4) from the eosinophils of healthy donors. CONCLUSIONS: The results indicated that PAF enhanced LTC4 formation by eosinophils obtained from asthmatic patients stimulated with the calcium ionophore A23187, but not those obtained from normal subjects. PMID:8711647

Synergistic effects of adenosine A1 and P2Y receptor stimulation on calcium mobilization and PKC translocation in DDT1 MF-2 cells.

PubMed

Fredholm, Bertil B; Assender, Jean W; Irenius, Eva; Kodama, Noriko; Saito, Naoaki

2003-06-01

1. The effect of adenosine analogues and of nucleotides, alone or in combination, on intracellular calcium, accumulation of inositol (1,4,5) trisphosphate (InsP3), and on activation of protein kinase C (PKC) was studied in DDT1 MF2 cells derived from a Syrian hamster myosarcoma. These cells were found to express mRNA for A1 and some as yet unidentified P2Y receptor(s). 2. Activation of either receptor type stimulated the production of InsP3 and raised intracellular calcium in DDT1 MF2 cells. Similarly, the A1 selective agonist N6-cyclopentyladenosine (CPA) increased PKC-dependent phosphorylation of the substrate MBP(4-14) and induced a PKC translocation to the plasma membrane as determined using [3H]-phorbol dibutyrate (PDBu) binding in DDT1 MF-2 cells. However, neither adenosine nor CPA induced a significant translocation of transiently transfected gamma-PKC-GFP from the cytosol to the cell membrane. In contrast to adenosine analogues, ATP and UTP also caused a rapid but transient translocation of gamma-PKC-GFP and activation of PKC. 3. Doses of the A1 agonist CPA and of ATP or UTP per se caused barely detectable increases in intracellular Ca2+ but when combined, they caused an almost maximal stimulation. Similarly, adenosine (0.6 microM) and UTP (or ATP, 2.5 microM), which per se caused no detectable translocation of either gamma- or epsilon-PKC-GFP, caused when combined a very clear-cut translocation of both PKC subforms, albeit with different time courses. These results show that simultaneous activation of P2Y and adenosine A1 receptors synergistically increases Ca2+ transients and translocation of PKC in DDT1 MF-2 cells. Since adenosine is rapidly formed by breakdown of extracellular ATP, such interactions may be biologically important.

Plant cell nucleolus as a hot spot for iron.

PubMed

Roschzttardtz, Hannetz; Grillet, Louis; Isaure, Marie-Pierre; Conéjéro, Geneviève; Ortega, Richard; Curie, Catherine; Mari, Stéphane

2011-08-12

Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.

Subpopulation-proteomics in prokaryotic populations.

PubMed

Jahn, Michael; Seifert, Jana; von Bergen, Martin; Schmid, Andreas; Bühler, Bruno; Müller, Susann

2013-02-01

Clonal microbial cells do not behave in an identical manner and form subpopulations during cultivation. Besides varying micro-environmental conditions, cell inherent features like cell cycle dependent localization and concentration of regulatory proteins as well as epigenetic properties are well accepted mechanisms creating cell heterogeneity. Another suspected reason is molecular noise on the transcriptional and translational level. A promising tool to unravel reasons for cell heterogeneity is the combination of cell sorting and subpopulation proteomics. This review summarizes recent developments in prokaryotic single-cell analytics and provides a workflow for selection of single cells, low cell number mass spectrometry, and proteomics evaluation. This approach is useful for understanding the dependency of individual cell decisions on inherent protein profiles. Copyright © 2012 Elsevier Ltd. All rights reserved.

Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

PubMed Central

Pavelin, Jon; Reynolds, Natalie; Chiweshe, Stephen; Wu, Guanming; Tiribassi, Rebecca; Grey, Finn

2013-01-01

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions. PMID:24385903

Solid oxide fuel cell systems for residential micro-combined heat and power in the UK: Key economic drivers

NASA Astrophysics Data System (ADS)

Hawkes, Adam; Leach, Matthew

The ability of combined heat and power (CHP) to meet residential heat and power demands efficiently offers potentially significant financial and environmental advantages over centralised power generation and heat-provision through natural-gas fired boilers. A solid oxide fuel cell (SOFC) can operate at high overall efficiencies (heat and power) of 80-90%, offering an improvement over centralised generation, which is often unable to utilise waste heat. This paper applies an equivalent annual cost (EAC) minimisation model to a residential solid oxide fuel cell CHP system to determine what the driving factors are behind investment in this technology. We explore the performance of a hypothetical SOFC system—representing expectations of near to medium term technology development—under present UK market conditions. We find that households with small to average energy demands do not benefit from installation of a SOFC micro-CHP system, but larger energy demands do benefit under these conditions. However, this result is sensitive to a number of factors including stack capital cost, energy import and export prices, and plant lifetime. The results for small and average dwellings are shown to reverse under an observed change in energy import prices, an increase in electricity export price, a decrease in stack capital costs, or an improvement in stack lifetime.

Support schemes and ownership structures - the policy context for fuel cell based micro-combined heat and power

NASA Astrophysics Data System (ADS)

Schroeder, Sascha Thorsten; Costa, Ana; Obé, Elisabeth

In recent years, fuel cell based micro-combined heat and power (mCHP) has received increasing attention due to its potential contribution to European energy policy goals, i.e., sustainability, competitiveness and security of supply. Besides technical advances, regulatory framework and ownership structures are of crucial importance in order to achieve greater diffusion of the technology in residential applications. This paper analyses the interplay of policy and ownership structures for the future deployment of mCHP. Furthermore, it regards the three country cases Denmark, France and Portugal. Firstly, the implications of different kinds of support schemes on investment risk and the diffusion of a technology are explained conceptually. Secondly, ownership arrangements are addressed. Then, a cross-country comparison on present support schemes for mCHP and competing technologies discusses the national implementation of European legislation in Denmark, France and Portugal. Finally, resulting implications for ownership arrangements on the choice of support scheme are explained. From a conceptual point of view, investment support, feed-in tariffs and price premiums are the most appropriate schemes for fuel cell mCHP. This can be used for improved analysis of operational strategies. The interaction of this plethora of elements necessitates careful balancing from a private- and socio-economic point of view.

4E analysis and multi objective optimization of a micro gas turbine and solid oxide fuel cell hybrid combined heat and power system

NASA Astrophysics Data System (ADS)

Sanaye, Sepehr; Katebi, Arash

2014-02-01

Energy, exergy, economic and environmental (4E) analysis and optimization of a hybrid solid oxide fuel cell and micro gas turbine (SOFC-MGT) system for use as combined generation of heat and power (CHP) is investigated in this paper. The hybrid system is modeled and performance related results are validated using available data in literature. Then a multi-objective optimization approach based on genetic algorithm is incorporated. Eight system design parameters are selected for the optimization procedure. System exergy efficiency and total cost rate (including capital or investment cost, operational cost and penalty cost of environmental emissions) are the two objectives. The effects of fuel unit cost, capital investment and system power output on optimum design parameters are also investigated. It is observed that the most sensitive and important design parameter in the hybrid system is fuel cell current density which has a significant effect on the balance between system cost and efficiency. The selected design point from the Pareto distribution of optimization results indicates a total system exergy efficiency of 60.7%, with estimated electrical energy cost 0.057 kW-1 h-1, and payback period of about 6.3 years for the investment.

A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E.; Pertsemlidis, Alexander; Du, Liqin

2014-01-01

Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy. PMID:24811707

Cellularizing hydrogel-based scaffolds to repair bone tissue: How to create a physiologically relevant micro-environment?

PubMed Central

Maisani, Mathieu; Pezzoli, Daniele; Chassande, Olivier; Mantovani, Diego

2017-01-01

Tissue engineering is a promising alternative to autografts or allografts for the regeneration of large bone defects. Cell-free biomaterials with different degrees of sophistication can be used for several therapeutic indications, to stimulate bone repair by the host tissue. However, when osteoprogenitors are not available in the damaged tissue, exogenous cells with an osteoblast differentiation potential must be provided. These cells should have the capacity to colonize the defect and to participate in the building of new bone tissue. To achieve this goal, cells must survive, remain in the defect site, eventually proliferate, and differentiate into mature osteoblasts. A critical issue for these engrafted cells is to be fed by oxygen and nutrients: the transient absence of a vascular network upon implantation is a major challenge for cells to survive in the site of implantation, and different strategies can be followed to promote cell survival under poor oxygen and nutrient supply and to promote rapid vascularization of the defect area. These strategies involve the use of scaffolds designed to create the appropriate micro-environment for cells to survive, proliferate, and differentiate in vitro and in vivo. Hydrogels are an eclectic class of materials that can be easily cellularized and provide effective, minimally invasive approaches to fill bone defects and favor bone tissue regeneration. Furthermore, by playing on their composition and processing, it is possible to obtain biocompatible systems with adequate chemical, biological, and mechanical properties. However, only a good combination of scaffold and cells, possibly with the aid of incorporated growth factors, can lead to successful results in bone regeneration. This review presents the strategies used to design cellularized hydrogel-based systems for bone regeneration, identifying the key parameters of the many different micro-environments created within hydrogels. PMID:28634532

Detection of viability of micro-algae cells by optofluidic hologram pattern.

PubMed

Wang, Junsheng; Yu, Xiaomei; Wang, Yanjuan; Pan, Xinxiang; Li, Dongqing

2018-03-01

A rapid detection of micro-algae activity is critical for analysis of ship ballast water. A new method for detecting micro-algae activity based on lens-free optofluidic holographic imaging is presented in this paper. A compact lens-free optofluidic holographic imaging device was developed. This device is mainly composed of a light source, a small through-hole, a light propagation module, a microfluidic chip, and an image acquisition and processing module. The excited light from the light source passes through a small hole to reach the surface of the micro-algae cells in the microfluidic chip, and a holographic image is formed by the diffraction light of surface of micro-algae cells. The relation between the characteristics in the hologram pattern and the activity of micro-algae cells was investigated by using this device. The characteristics of the hologram pattern were extracted to represent the activity of micro-algae cells. To demonstrate the accuracy of the presented method and device, four species of micro-algae cells were employed as the test samples and the comparison experiments between the alive and dead cells of four species of micro-algae were conducted. The results show that the developed method and device can determine live/dead microalgae cells accurately.

Micro/nano-fabrication technologies for cell biology.

PubMed

Qian, Tongcheng; Wang, Yingxiao

2010-10-01

Micro/nano-fabrication techniques, such as soft lithography and electrospinning, have been well-developed and widely applied in many research fields in the past decade. Due to the low costs and simple procedures, these techniques have become important and popular for biological studies. In this review, we focus on the studies integrating micro/nano-fabrication work to elucidate the molecular mechanism of signaling transduction in cell biology. We first describe different micro/nano-fabrication technologies, including techniques generating three-dimensional scaffolds for tissue engineering. We then introduce the application of these technologies in manipulating the physical or chemical micro/nano-environment to regulate the cellular behavior and response, such as cell life and death, differentiation, proliferation, and cell migration. Recent advancement in integrating the micro/nano-technologies and live cell imaging are also discussed. Finally, potential schemes in cell biology involving micro/nano-fabrication technologies are proposed to provide perspectives on the future research activities.

Micro/nano-fabrication technologies for cell biology

PubMed Central

Qian, Tongcheng

2012-01-01

Micro/nano-fabrication techniques, such as soft lithography and electrospinning, have been well-developed and widely applied in many research fields in the past decade. Due to the low costs and simple procedures, these techniques have become important and popular for biological studies. In this review, we focus on the studies integrating micro/nano-fabrication work to elucidate the molecular mechanism of signaling transduction in cell biology. We first describe different micro/nano-fabrication technologies, including techniques generating three-dimensional scaffolds for tissue engineering. We then introduce the application of these technologies in manipulating the physical or chemical micro/nano-environment to regulate the cellular behavior and response, such as cell life and death, differentiation, proliferation, and cell migration. Recent advancement in integrating the micro/nano-technologies and live cell imaging are also discussed. Finally, potential schemes in cell biology involving micro/nano-fabrication technologies are proposed to provide perspectives on the future research activities. PMID:20490938

The IL-4/STAT6 signaling axis establishes a conserved microRNA signature in human and mouse macrophages regulating cell survival via miR-342-3p.

PubMed

Czimmerer, Zsolt; Varga, Tamas; Kiss, Mate; Vázquez, Cesaré Ovando; Doan-Xuan, Quang Minh; Rückerl, Dominik; Tattikota, Sudhir Gopal; Yan, Xin; Nagy, Zsuzsanna S; Daniel, Bence; Poliska, Szilard; Horvath, Attila; Nagy, Gergely; Varallyay, Eva; Poy, Matthew N; Allen, Judith E; Bacso, Zsolt; Abreu-Goodger, Cei; Nagy, Laszlo

2016-05-31

IL-4-driven alternative macrophage activation and proliferation are characteristic features of both antihelminthic immune responses and wound healing in contrast to classical macrophage activation, which primarily occurs during inflammatory responses. The signaling pathways defining the genome-wide microRNA expression profile as well as the cellular functions controlled by microRNAs during alternative macrophage activation are largely unknown. Hence, in the current work we examined the regulation and function of IL-4-regulated microRNAs in human and mouse alternative macrophage activation. We utilized microarray-based microRNA profiling to detect the dynamic expression changes during human monocyte-macrophage differentiation and IL-4-mediated alternative macrophage activation. The expression changes and upstream regulatory pathways of selected microRNAs were further investigated in human and mouse in vitro and in vivo models of alternative macrophage activation by integrating small RNA-seq, ChIP-seq, ChIP-quantitative PCR, and gene expression data. MicroRNA-controlled gene networks and corresponding functions were identified using a combination of transcriptomic, bioinformatic, and functional approaches. The IL-4-controlled microRNA expression pattern was identified in models of human and mouse alternative macrophage activation. IL-4-dependent induction of miR-342-3p and repression of miR-99b along with miR-125a-5p occurred in both human and murine macrophages in vitro. In addition, a similar expression pattern was observed in peritoneal macrophages of Brugia malayi nematode-implanted mice in vivo. By using IL4Rα- and STAT6-deficient macrophages, we were able to show that IL-4-dependent regulation of miR-342-3p, miR-99b, and miR-125a-5p is mediated by the IL-4Rα-STAT6 signaling pathway. The combination of gene expression studies and chromatin immunoprecipitation experiments demonstrated that both miR-342-3p and its host gene, EVL, are coregulated directly by STAT6. Finally, we found that miR-342-3p is capable of controlling macrophage survival through targeting an anti-apoptotic gene network including Bcl2l1. Our findings identify a conserved IL-4/STAT6-regulated microRNA signature in alternatively activated human and mouse macrophages. Moreover, our study indicates that miR-342-3p likely plays a pro-apoptotic role in such cells, thereby providing a negative feedback arm to IL-4-dependent macrophage proliferation.

What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast

PubMed Central

Lee, Cheryl Q.E.; Gardner, Lucy; Turco, Margherita; Zhao, Nancy; Murray, Matthew J.; Coleman, Nicholas; Rossant, Janet; Hemberger, Myriam; Moffett, Ashley

2016-01-01

Summary Controversy surrounds reports describing the derivation of human trophoblast cells from placentas and embryonic stem cells (ESC), partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. We have selected criteria that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs (miRNAs) from the chromosome 19 miRNA cluster (C19MC). We tested these criteria on cells previously reported to show some phenotypic characteristics of trophoblast: bone morphogenetic protein (BMP)-treated human ESC and 2102Ep, an embryonal carcinoma cell line. Both cell types only show some, but not all, of the four trophoblast criteria. Thus, BMP-treated human ESC have not fully differentiated to trophoblast. Our study identifies a robust panel, including both protein and non-protein-coding markers that, in combination, can be used to reliably define cells as characteristic of early trophoblast. PMID:26862703

MEMS resonant load cells for micro-mechanical test frames: feasibility study and optimal design

NASA Astrophysics Data System (ADS)

Torrents, A.; Azgin, K.; Godfrey, S. W.; Topalli, E. S.; Akin, T.; Valdevit, L.

2010-12-01

This paper presents the design, optimization and manufacturing of a novel micro-fabricated load cell based on a double-ended tuning fork. The device geometry and operating voltages are optimized for maximum force resolution and range, subject to a number of manufacturing and electromechanical constraints. All optimizations are enabled by analytical modeling (verified by selected finite elements analyses) coupled with an efficient C++ code based on the particle swarm optimization algorithm. This assessment indicates that force resolutions of ~0.5-10 nN are feasible in vacuum (~1-50 mTorr), with force ranges as large as 1 N. Importantly, the optimal design for vacuum operation is independent of the desired range, ensuring versatility. Experimental verifications on a sub-optimal device fabricated using silicon-on-glass technology demonstrate a resolution of ~23 nN at a vacuum level of ~50 mTorr. The device demonstrated in this article will be integrated in a hybrid micro-mechanical test frame for unprecedented combinations of force resolution and range, displacement resolution and range, optical (or SEM) access to the sample, versatility and cost.

Overexpression of microRNA-194 suppresses the epithelial-mesenchymal transition in targeting stem cell transcription factor Sox3 in endometrial carcinoma stem cells.

PubMed

Gong, Baolan; Yue, Yan; Wang, Renxiao; Zhang, Yi; Jin, Quanfang; Zhou, Xi

2017-06-01

The epithelial-mesenchymal transition is the key process driving cancer metastasis. MicroRNA-194 inhibits epithelial-mesenchymal transition in several cancers and its downregulation indicates a poor prognosis in human endometrial carcinoma. Self-renewal factor Sox3 induces epithelial-mesenchymal transition at gastrulation and is also involved epithelial-mesenchymal transition in several cancers. We intended to determine the roles of Sox3 in inducing epithelial-mesenchymal transition in endometrial cancer stem cells and the possible role of microRNA-194 in controlling Sox3 expression. Firstly, we found that Sox3 and microRNA-194 expressions were associated with the status of endometrial cancer stem cells in a panel of endometrial carcinoma tissue, the CD133+ cell was higher in tumorsphere than in differentiated cells, and overexpression of microRNA-194 would decrease CD133+ cell expression. Silencing of Sox3 in endometrial cancer stem cell upregulated the epithelial marker E-cadherin, downregulated the mesenchymal marker vimentin, and significantly reduced cell invasion in vitro; overexpression of Sox3 reversed these phenotypes. Furthermore, we discovered that the expression of Sox3 was suppressed by microRNA-194 through direct binding to the Sox3 3'-untranslated region. Ectopic expression of microRNA-194 in endometrial cancer stem cells induced a mesenchymal-epithelial transition by restoring E-cadherin expression, decreasing vimentin expression, and inhibiting cell invasion in vitro. Moreover, overexpression of microRNA-194 inhibited endometrial cancer stem cell invasion or metastasis in vivo by injection of adenovirus microRNA-194. These findings demonstrate the novel mechanism by which Sox3 contributes to endometrial cancer stem cell invasion and suggest that repression of Sox3 by microRNA-194 may have therapeutic potential to suppress endometrial carcinoma metastasis. The cancer stem cell marker, CD133, might be the surface marker of endometrial cancer stem cell.

Surface characteristics and biocompatibility of cranioplasty titanium implants following different surface treatments.

PubMed

Hatamleh, Muhanad M; Wu, Xiaohong; Alnazzawi, Ahmad; Watson, Jason; Watts, David

2018-04-01

Surface and mechanical properties of titanium alloys are integral for their use in restoring bone defects of skull and face regions. These properties are affected by the method of constructing and surface treatment of the titanium implant. This study aimed to investigate the effects of titanium finishing protocols on the surface morphology, hardness and biocompatibility of TiAl6V4. Square shaped TiAl6V4 specimens (ASTM F68) (10×10×0.5mm) were divided into seven groups of different surface treatments (n=10). The treatments included mechanical polishing, sandblasting with AL 2 O 3 (50μm), immersion in different acids, and/or electro-chemical anodization. Weight loss %; 3D micro-roughness; Knoop micro-hardness, and osteoblast cell attachment and proliferation (after 3 days) were determined for each specimen. Data was analysed using one way ANOVA and Dunett T3 post-hoc tests, and t-test (p<0.05). Weight loss % was in the range of 1.70-5.60 as mechanical polishing produced the highest weight loss, followed by sandblasting, and combined protocol of mechanical polishing and acid treatment (p<0.05). Micro-roughness values (μm) were in the range of 2.81-16.68. It was the highest for control specimens (p<0.05), and smoothest surfaces after combined mechanical polishing and acid treatment; or after electro-chemical treatment (p<0.05). Micro-hardness values (MPa) ranged 170.90-442.15 as sandblasting with/without acid treatment caused statically significantly the highest values (p<0.05) while control and mechanically polished specimens had the lowest values (p<0.05). All treatments produced equally biocompatible surfaces (p>0.05) after 1h or 3 days. Furthermore, osteoblast cell proliferation statistically significantly increased after 3days among each surface treatment (p<0.05). Different finishing treatments have variable effect on cranioplasty titanium surface loss, micro-roughness and micro-hardness but constant improved biocompatibility effect. Electro-chemical treatment caused less material loss and produced biocompatible smoothest surface of comparable hardness; hence it can be suitable for cranioplasty titanium surface finishing. Copyright © 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Combination with third-generation bisphosphonate (YM529) and interferon-alpha can inhibit the progression of established bone renal cell carcinoma.

PubMed

Kurabayashi, Atsushi; Inoue, Keiji; Fukuhara, Hideo; Karashima, Takashi; Fukata, Satoshi; Kawada, Chiaki; Shuin, Taro; Furihata, Mutsuo

2015-08-01

The aim of this study was to investigate whether the third-generation nitrogen-containing bisphosphonate (YM529) can inhibit the progression of established bone renal cell carcinoma (RCC) and to elucidate its mechanism. Antiproliferative effect and apoptosis induction of RCC cells and mouse osteoclasts by YM529 and/or interferon-alpha (IFN-α) were evaluated in vitro using cell counting and in vivo using soft X-ray, the TUNEL method and tartrate-resistant acid phosphatase stain. For the in vivo study, male athymic BALB/cA Jc1-nu nude mice bearing human RCC cell line RBM1-IT4 cells were treated with YM529 and/or IFN-α. The biological activity of osteoclasts was evaluated using the pit formation assay. The antiangiogenetic effect by YM529 and/or IFN-α was analyzed using micro-vessel density and in situ mRNA hybridization. Osteoclast number in bone tumors was decreased in YM529-treated mouse. YM529 also inhibited osteoclast activity and proliferation in vitro, whereas basic fibroblast growth factor expressions and micro-vessel density within tumors were inhibited by IFN-α. Neither YM529 nor IFN-α alone significantly inhibited the growth of established bone metastatic tumors. Combined treatment with YM529 and IFN-α may be beneficial in patients with human RCC bone metastasis. Their effects are mediated by osteoclast recruitment inhibition and inactivation by YM529 and antiangiogenesis by IFN-α. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

The aquatic hyphomycete Heliscus lugdunensis protects its hyphae tip cells from cadmium: A micro X-ray fluorescence and X-ray absorption near edge structure spectroscopy study

NASA Astrophysics Data System (ADS)

Isaure, Marie-Pierre; Leyh, Benjamin; Salomé, Murielle; Krauss, Gerd-Joachim; Schaumlöffel, Dirk; Dobritzsch, Dirk

2017-11-01

Aquatic fungi can be used to evaluate the functioning of natural ecosystems. Heliscus lugdunensis is an early colonizer of allochthone leafs. Since this aquatic hyphomycete is able to develop in metal contaminated habitats and tolerates cadmium, it appears to be a good candidate to investigate adaptation to metal pollution. This study aimed at examining the sequestration of Cd in the hyphae of H. lugdunensis, and particularly the role of the tip cells. For that, H. lugdunensis growth was evaluated under various Cd concentrations, and a combination of synchrotron micro X-ray fluorescence and X-ray absorption near edge structure spectroscopy was carried out to determine the compartments of Cd accumulation and the Cd chemical species, respectively. Results showed that the hyphal tip cells were depleted in Cd, and that the metal was stored in older cells. Cd was mainly associated with sulfur ligands and to a lesser extent bound to phosphates and carboxyl/hydroxyl groups from cell wall and/or organic acids. Finally, the aquatic fungus was able to maintain the tip cell as a functional system, thus allowing the colonization of contaminated environments.

MicroRNA-300 targets hypoxia inducible factor-3 alpha to inhibit tumorigenesis of human non-small cell lung cancer.

PubMed

Zhang, Y; Guo, Y; Yang, C; Zhang, S; Zhu, X; Cao, L; Nie, W; Yu, H

2017-01-01

Non-small cell lung cancer (NSCLC) is one of the most deadly human cancers. MicroRNA-300 acts as both tumor promoter and suppressor in different types of cancer. Here, we try to identify the function of microRNA-300 in human NSCLC. We compared MicroRNA-300 levels between tumor tissues versus paired adjacent non-tumor lung tissues from NSCLC patients, and in NSCLC versus normal lung cell lines. Effects of microRNA-300 on cell proliferation, invasion and migration were examined in vitro, and on tumor growth in vivo using a xenograft mouse model. Potential mRNA targets of microRNA-300 were predicted and underlying mechanism was explored. MicroRNA-300 expression was lower in both NSCLC tissues and cell lines. Overexpression of microRNA-300 inhibited proliferation, invasion and migration of NSCLC cells in vitro, and tumor growth in vivo. MicroRNA-300 could directly bind to the 3'-UTR of hypoxia inducible factor-3 alpha (HIF3α) mRNA, and inhibit both its mRNA and protein expressions. Restoring HIF3α expression could rescue the inhibitory effects of microRNA-300 on tumorigenesis of NSCLC both in vitro and in vivo. MicroRNA-300 is a tumor suppressor microRNA in NSCLC by downregulating HIF3α expression. Both microRNA-300 and HIF3α may serve as potential therapeutic targets in NSCLC treatment.

Four MicroRNAs Promote Prostate Cell Proliferation with Regulation of PTEN and Its Downstream Signals In Vitro

PubMed Central

Xue, Jing-lun; Chen, Jin-zhong

2013-01-01

Background Phosphatase and tensin homologue (PTEN), as a tumor suppressor, plays vital roles in tumorigenesis and progression of prostate cancer. However, the mechanisms of PTEN regulation still need further investigation. We here report that a combination of four microRNAs (miR-19b, miR-23b, miR-26a and miR-92a) promotes prostate cell proliferation by regulating PTEN and its downstream signals in vitro. Methodology/Principal Findings We found that the four microRNAs (miRNAs) could effectively suppress PTEN expression by directly interacting with its 3’ UTR in prostate epithelial and cancer cells. Under-expression of the four miRNAs by antisense neutralization up-regulates PTEN expression, while overexpression of the four miRNAs accelerates epithelial and prostate cancer cell proliferation. Furthermore, the expression of the four miRNAs could, singly or jointly, alter the expression of the key components in the phosphoinositide 3-kinase (PI3K)/Akt pathway, including PIK3CA, PIK3CD, PIK3R1 and Akt, along with their downstream signal, cyclin D1. Conclusions These results suggested that the four miRNAs could promote prostate cancer cell proliferation by co-regulating the expression of PTEN, PI3K/Akt pathway and cyclin D1 in vitro. These findings increase understanding of the molecular mechanisms of prostate carcinogenesis and progression, even provide valuable insights into the diagnosis, prognosis, and rational design of novel therapeutics for prostate cancer. PMID:24098737

MicroRNA-99 Family Targets AKT/mTOR Signaling Pathway in Dermal Wound Healing

PubMed Central

Chen, Dan; Fang, Zong Juan; Zhao, Yan; Dragas, Dragan; Dai, Yang; Marucha, Phillip T.; Zhou, Xiaofeng

2013-01-01

Recent studies suggest that microRNAs play important roles in dermal wound healing and microRNA deregulation has been linked with impaired wound repair. Here, using a mouse experimental wound healing model, we identified a panel of 63 differentially expressed microRNAs during dermal wound healing, including members of miR-99 family (miR-99a, miR-99b, miR-100). We further demonstrated that miR-99 family members regulate cell proliferation, cell migration, and AKT/mTOR signaling. Combined experimental and bioinformatics analyses revealed that miR-99 family members regulate AKT/mTOR signaling by targeting multiple genes, including known target genes (e.g., IGF1R, mTOR) and a new target (AKT1). The effects of miR-99 family members on the expression of IGF1R, mTOR and AKT1 were validated at both the mRNA and protein levels. Two adjacent miR-99 family targeting sites were identified in the 3′-UTR of the AKT1 mRNA. The direct interaction of miR-100 with these targeting sites was confirmed using luciferase reporter assays. The microRNA-100-directed recruitment of AKT1 mRNA to the RNAi-induced silencing complex (RISC) was confirmed by a ribonucleoprotein-IP assay. In summary, we identified a panel of differentially expressed microRNAs which may play important roles in wound healing. We provide evidence that miR-99 family members contribute to wound healing by regulating the AKT/mTOR signaling. PMID:23724047

MicroRNA-99 family targets AKT/mTOR signaling pathway in dermal wound healing.

PubMed

Jin, Yi; Tymen, Stéphanie D; Chen, Dan; Fang, Zong Juan; Zhao, Yan; Dragas, Dragan; Dai, Yang; Marucha, Phillip T; Zhou, Xiaofeng

2013-01-01

Recent studies suggest that microRNAs play important roles in dermal wound healing and microRNA deregulation has been linked with impaired wound repair. Here, using a mouse experimental wound healing model, we identified a panel of 63 differentially expressed microRNAs during dermal wound healing, including members of miR-99 family (miR-99a, miR-99b, miR-100). We further demonstrated that miR-99 family members regulate cell proliferation, cell migration, and AKT/mTOR signaling. Combined experimental and bioinformatics analyses revealed that miR-99 family members regulate AKT/mTOR signaling by targeting multiple genes, including known target genes (e.g., IGF1R, mTOR) and a new target (AKT1). The effects of miR-99 family members on the expression of IGF1R, mTOR and AKT1 were validated at both the mRNA and protein levels. Two adjacent miR-99 family targeting sites were identified in the 3'-UTR of the AKT1 mRNA. The direct interaction of miR-100 with these targeting sites was confirmed using luciferase reporter assays. The microRNA-100-directed recruitment of AKT1 mRNA to the RNAi-induced silencing complex (RISC) was confirmed by a ribonucleoprotein-IP assay. In summary, we identified a panel of differentially expressed microRNAs which may play important roles in wound healing. We provide evidence that miR-99 family members contribute to wound healing by regulating the AKT/mTOR signaling.

Knockdown of miR-27a sensitizes colorectal cancer stem cells to TRAIL by promoting the formation of Apaf-1-caspase-9 complex

PubMed Central

Zhang, Rui; Xu, Jian; Zhao, Jian; Bai, Jinghui

2017-01-01

MicroRNAs have been proved to participate in multiple biological processes in cancers. For developing resistance to cytotoxic drug, cancer cells, especially the cancer stem cells, usually change their microRNA expression profile to survive in hostile environments. In the present study, we found that expression of microRNA-27a was increased in colorectal cancer stem cells. High level of microRNA-27a was indicated to induce the resistance to TNF-related apoptosis-inducing ligand (TRAIL). Knockdown of microRNA-27a resensitized colorectal cancer stem cells to TRAIL-induced cell death. Mechanically, the gene of Apaf-1, which is associated with the mitochondrial apoptosis, was demonstrated to be the target of microRNA-27a in colorectal cancer stem cells. Knockdown of microRNA-27a increased the expression level of Apaf-1, thus enhancing the formation of Apaf-1-caspase-9 complex and subsequently promoting the TRAIL-induced apoptosis in colorectal cancer stem cells. These findings suggested that knockdown of microRNA-27a in colorectal cancer stem cells by the specific antioligonucleotides was potential to reverse the chemoresistance to TRAIL. It may represent a novel therapeutic strategy for treating the colorectal cancer more effectively. PMID:28423356

Knockdown of miR-27a sensitizes colorectal cancer stem cells to TRAIL by promoting the formation of Apaf-1-caspase-9 complex.

PubMed

Zhang, Rui; Xu, Jian; Zhao, Jian; Bai, Jinghui

2017-07-11

MicroRNAs have been proved to participate in multiple biological processes in cancers. For developing resistance to cytotoxic drug, cancer cells, especially the cancer stem cells, usually change their microRNA expression profile to survive in hostile environments. In the present study, we found that expression of microRNA-27a was increased in colorectal cancer stem cells. High level of microRNA-27a was indicated to induce the resistance to TNF-related apoptosis-inducing ligand (TRAIL). Knockdown of microRNA-27a resensitized colorectal cancer stem cells to TRAIL-induced cell death. Mechanically, the gene of Apaf-1, which is associated with the mitochondrial apoptosis, was demonstrated to be the target of microRNA-27a in colorectal cancer stem cells. Knockdown of microRNA-27a increased the expression level of Apaf-1, thus enhancing the formation of Apaf-1-caspase-9 complex and subsequently promoting the TRAIL-induced apoptosis in colorectal cancer stem cells. These findings suggested that knockdown of microRNA-27a in colorectal cancer stem cells by the specific antioligonucleotides was potential to reverse the chemoresistance to TRAIL. It may represent a novel therapeutic strategy for treating the colorectal cancer more effectively.

Refractive multiple optical tweezers for parallel biochemical analysis in micro-fluidics

NASA Astrophysics Data System (ADS)

Merenda, Fabrice; Rohner, Johann; Pascoal, Pedro; Fournier, Jean-Marc; Vogel, Horst; Salathé, René-Paul

2007-02-01

We present a multiple laser tweezers system based on refractive optics. The system produces an array of 100 optical traps thanks to a refractive microlens array, whose focal plane is imaged into the focal plane of a high-NA microscope objective. This refractive multi-tweezers system is combined to micro-fluidics, aiming at performing simultaneous biochemical reactions on ensembles of free floating objects. Micro-fluidics allows both transporting the particles to the trapping area, and conveying biochemical reagents to the trapped particles. Parallel trapping in micro-fluidics is achieved with polystyrene beads as well as with native vesicles produced from mammalian cells. The traps can hold objects against fluid flows exceeding 100 micrometers per second. Parallel fluorescence excitation and detection on the ensemble of trapped particles is also demonstrated. Additionally, the system is capable of selectively and individually releasing particles from the tweezers array using a complementary steerable laser beam. Strategies for high-yield particle capture and individual particle release in a micro-fluidic environment are discussed. A comparison with diffractive optical tweezers enhances the pros and cons of refractive systems.

MiR-21 suppresses the anticancer activities of curcumin by targeting PTEN gene in human non-small cell lung cancer A549 cells.

PubMed

Zhang, W; Bai, W; Zhang, W

2014-08-01

Curcumin, a natural phytochemical, exhibits potent anticancer activities. Here, we sought to determine the molecular mechanisms underlying the cytotoxic effects of curcumin against human non-small cell lung cancer (NSCLC) cells. MTT assay and annexin-V/PI staining were used to analyze the effects of curcumin on the proliferation and apoptosis of A549 cells. The expression of microRNA-21 in curcumin-treated A549 cells was measured by quantitative real-time polymerase chain reaction assay. The protein level of phosphatase and tensin homolog (PTEN), a putative target of microRNA-21, was determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA was performed to modulate the expression of microRNA-21 and PTEN under the treatment of curcumin. Curcumin at 20-40 μM inhibited cell proliferation and induced apoptosis in A549 cells. Curcumin treatment produced a dose-dependent and significant (P < 0.05) suppression of microRNA-21 expression, compared to untreated A549 cells. Moreover, the protein level of PTEN, a putative target of microRNA-21, was significantly elevated in curcumin-treated A549 cells, as determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA significantly (P < 0.05) reversed the growth suppression and apoptosis induction by curcumin, compared to corresponding controls. Our data suggest a novel molecular mechanism in which inhibition of microRNA-21 and upregulation of PTEN mediate the anticancer activities of curcumin in NSCLC cells. Suppression of microRNA-21 may thus have therapeutic benefits against this malignancy.

Intracellular and Extracellular Recording of Spontaneous Action Potentials in Mammalian Neurons and Cardiac Cells with 3D Plasmonic Nanoelectrodes.

PubMed

Dipalo, Michele; Amin, Hayder; Lovato, Laura; Moia, Fabio; Caprettini, Valeria; Messina, Gabriele C; Tantussi, Francesco; Berdondini, Luca; De Angelis, Francesco

2017-06-14

Three-dimensional vertical micro- and nanostructures can enhance the signal quality of multielectrode arrays and promise to become the prime methodology for the investigation of large networks of electrogenic cells. So far, access to the intracellular environment has been obtained via spontaneous poration, electroporation, or by surface functionalization of the micro/nanostructures; however, these methods still suffer from some limitations due to their intrinsic characteristics that limit their widespread use. Here, we demonstrate the ability to continuously record both extracellular and intracellular-like action potentials at each electrode site in spontaneously active mammalian neurons and HL-1 cardiac-derived cells via the combination of vertical nanoelectrodes with plasmonic optoporation. We demonstrate long-term and stable recordings with a very good signal-to-noise ratio. Additionally, plasmonic optoporation does not perturb the spontaneous electrical activity; it permits continuous recording even during the poration process and can regulate extracellular and intracellular contributions by means of partial cellular poration.

Fermentation Results in Quantitative Changes in Milk-Derived Exosomes and Different Effects on Cell Growth and Survival.

PubMed

Yu, Siran; Zhao, Zhehao; Sun, Liming; Li, Ping

2017-02-15

The discovery of microRNAs encapsulated in milk-derived exosomes has revealed stability under extreme conditions reflecting the protection of membranes. We attempted to determine the variations in nanoparticles derived from milk after fermentation, and provide evidence to determine the effects of these exosomes on cells with potential bioactivity. Using scanning electron microscopy and dynamic light scattering, we compared the morphology and particle size distribution of exosomes from yogurt fermented with three different combinations of strains with those from raw milk. The protein content of the exosome was significantly reduced in fermented milk. The cycle threshold showed that the expression of miR-29b and miR-21 was relatively high in raw milk, indicating a loss of microRNA after fermentation. Milk-derived exosomes could promote cell growth and activate the mitogen-activated protein kinase pathway. These findings demonstrated biological functions in milk exosomes and provided new insight into the nutrient composition of dairy products.

Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture.

PubMed

Störmer, Elke; von Moltke, Lisa L; Perloff, Michael D; Greenblatt, David J

2002-07-01

This study investigated the effects of the non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) nevirapine (NVR), efavirenz (EFV), and delavirdine (DLV) on P-glycoprotein (P-gp) activity and expression to anticipate P-gp related drug-drug interactions associated with combination therapy. NNRTIs were evaluated as P-gp substrates by measuring differential transport across Caco-2 cell monolayers. Inhibition of P-gp mediated rhodaminel23 (Rh123) transport in Caco-2 cells was used to assess P-gp inhibition by NNRTIs. Induction of P-gp expression and activity in LS180V cells following 3-day exposure to NNRTIs was measured by western blot analysis and cellular Rh123 uptake, respectively. The NNRTIs showed no differential transport between the basolateral to apical and apical to basolateral direction. NNRTI transport in either direction was not affected by the P-gp inhibitor verapamil. DLV inhibited Rh123 transport, causing a reduction to 15% of control at 100 microM (IC50 = 30 microM). NVR caused a concentration-dependent induction of P-gp expression in LS180V cells resulting in a 3.5-fold increase in immunoreactive P-gp at 100 microM NVR. Induction attributable to EFV and DLV was quantitatively smaller. NVR significantly reduced cellular uptake of Rh123 into LS180V cells, indicating increased drug efflux due to induced P-gp activity; effects of EFV and DLV were smaller. Acute DLV treatment of LS180V cells previously induced with NVR or ritonavir did not reverse the decreased Rh123 cell accumulation. NNRTIs show differential effects on P-gp activity and expression in vitro. Clinical studies are required to elucidate the clinical importance of potential drug interactions.

miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells.

PubMed

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A; Voorhoeve, P Mathijs

2012-05-01

MicroRNA-mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3' UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3' UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3' UTR-mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs.

miR-Sens—a retroviral dual-luciferase reporter to detect microRNA activity in primary cells

PubMed Central

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A.; Voorhoeve, P. Mathijs

2012-01-01

MicroRNA–mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3′ UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3′ UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3′ UTR–mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs. PMID:22417692

Optical and electrical interfacing technologies for living cell bio-chips.

PubMed

Shacham-Diamand, Y; Belkin, S; Rishpon, J; Elad, T; Melamed, S; Biran, A; Yagur-Kroll, S; Almog, R; Daniel, R; Ben-Yoav, H; Rabner, A; Vernick, S; Elman, N; Popovtzer, R

2010-06-01

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by micro-system-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such whole-cell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the micro-electronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

Life on magnets: stem cell networking on micro-magnet arrays.

PubMed

Zablotskii, Vitalii; Dejneka, Alexandr; Kubinová, Šárka; Le-Roy, Damien; Dumas-Bouchiat, Frédéric; Givord, Dominique; Dempsey, Nora M; Syková, Eva

2013-01-01

Interactions between a micro-magnet array and living cells may guide the establishment of cell networks due to the cellular response to a magnetic field. To manipulate mesenchymal stem cells free of magnetic nanoparticles by a high magnetic field gradient, we used high quality micro-patterned NdFeB films around which the stray field's value and direction drastically change across the cell body. Such micro-magnet arrays coated with parylene produce high magnetic field gradients that affect the cells in two main ways: i) causing cell migration and adherence to a covered magnetic surface and ii) elongating the cells in the directions parallel to the edges of the micro-magnet. To explain these effects, three putative mechanisms that incorporate both physical and biological factors influencing the cells are suggested. It is shown that the static high magnetic field gradient generated by the micro-magnet arrays are capable of assisting cell migration to those areas with the strongest magnetic field gradient, thereby allowing the build up of tunable interconnected stem cell networks, which is an elegant route for tissue engineering and regenerative medicine.

Life on Magnets: Stem Cell Networking on Micro-Magnet Arrays

PubMed Central

Zablotskii, Vitalii; Dejneka, Alexandr; Kubinová, Šárka; Le-Roy, Damien; Dumas-Bouchiat, Frédéric; Givord, Dominique; Dempsey, Nora M.; Syková, Eva

2013-01-01

Interactions between a micro-magnet array and living cells may guide the establishment of cell networks due to the cellular response to a magnetic field. To manipulate mesenchymal stem cells free of magnetic nanoparticles by a high magnetic field gradient, we used high quality micro-patterned NdFeB films around which the stray field’s value and direction drastically change across the cell body. Such micro-magnet arrays coated with parylene produce high magnetic field gradients that affect the cells in two main ways: i) causing cell migration and adherence to a covered magnetic surface and ii) elongating the cells in the directions parallel to the edges of the micro-magnet. To explain these effects, three putative mechanisms that incorporate both physical and biological factors influencing the cells are suggested. It is shown that the static high magnetic field gradient generated by the micro-magnet arrays are capable of assisting cell migration to those areas with the strongest magnetic field gradient, thereby allowing the build up of tunable interconnected stem cell networks, which is an elegant route for tissue engineering and regenerative medicine. PMID:23936425

“Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca2+ fluoroimaging”

PubMed Central

Kobayashi, Takuma; Haruta, Makito; Sasagawa, Kiyotaka; Matsumata, Miho; Eizumi, Kawori; Kitsumoto, Chikara; Motoyama, Mayumi; Maezawa, Yasuyo; Ohta, Yasumi; Noda, Toshihiko; Tokuda, Takashi; Ishikawa, Yasuyuki; Ohta, Jun

2016-01-01

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca2+ indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca2+ dynamics of neural cells were visualized simultaneously by fluorescence imaging. PMID:26878910

Trapping and dynamic manipulation of polystyrene beads mimicking circulating tumor cells using targeted magnetic/photoacoustic contrast agents

NASA Astrophysics Data System (ADS)

Wei, Chen-Wei; Xia, Jinjun; Pelivanov, Ivan; Hu, Xiaoge; Gao, Xiaohu; O'Donnell, Matthew

2012-10-01

Results on magnetically trapping and manipulating micro-scale beads circulating in a flow field mimicking metastatic cancer cells in human peripheral vessels are presented. Composite contrast agents combining magneto-sensitive nanospheres and highly optical absorptive gold nanorods were conjugated to micro-scale polystyrene beads. To efficiently trap the targeted objects in a fast stream, a dual magnet system consisting of two flat magnets to magnetize (polarize) the contrast agent and an array of cone magnets producing a sharp gradient field to trap the magnetized contrast agent was designed and constructed. A water-ink solution with an optical absorption coefficient of 10 cm-1 was used to mimic the optical absorption of blood. Magnetomotive photoacoustic imaging helped visualize bead trapping, dynamic manipulation of trapped beads in a flow field, and the subtraction of stationary background signals insensitive to the magnetic field. The results show that trafficking micro-scale objects can be effectively trapped in a stream with a flow rate up to 12 ml/min and the background can be significantly (greater than 15 dB) suppressed. It makes the proposed method very promising for sensitive detection of rare circulating tumor cells within high flow vessels with a highly absorptive optical background.

“Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca2+ fluoroimaging”

NASA Astrophysics Data System (ADS)

Kobayashi, Takuma; Haruta, Makito; Sasagawa, Kiyotaka; Matsumata, Miho; Eizumi, Kawori; Kitsumoto, Chikara; Motoyama, Mayumi; Maezawa, Yasuyo; Ohta, Yasumi; Noda, Toshihiko; Tokuda, Takashi; Ishikawa, Yasuyuki; Ohta, Jun

2016-02-01

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca2+ indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca2+ dynamics of neural cells were visualized simultaneously by fluorescence imaging.

Increasing the intracellular availability of all-trans retinoic acid in neuroblastoma cells.

PubMed

Armstrong, J L; Ruiz, M; Boddy, A V; Redfern, C P F; Pearson, A D J; Veal, G J

2005-02-28

Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 microM) or R116010 (1 or 10 microM) in combination with either 10 microM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma.

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

PubMed

Yamada, Mitsuhiro; Kubo, Hiroshi; Ota, Chiharu; Takahashi, Toru; Tando, Yukiko; Suzuki, Takaya; Fujino, Naoya; Makiguchi, Tomonori; Takagi, Kiyoshi; Suzuki, Takashi; Ichinose, Masakazu

2013-09-24

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition. Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.

Plasmofluidics: Merging Light and Fluids at the Micro-/Nano-Scale

PubMed Central

Wang, Mingsong; Zhao, Chenglong; Miao, Xiaoyu; Zhao, Yanhui; Rufo, Joseph

2016-01-01

Plasmofluidics is the synergistic integration of plasmonics and micro/nano fluidics in devices and applications in order to enhance performance. There has been significant progress in the emerging field of plasmofluidics in recent years. By utilizing the capability of plasmonics to manipulate light at the nanoscale, combined with the unique optical properties of fluids, and precise manipulation via micro/nano fluidics, plasmofluidic technologies enable innovations in lab-on-a-chip systems, reconfigurable photonic devices, optical sensing, imaging, and spectroscopy. In this review article, we examine and categorize the most recent advances in plasmofluidics into plasmon-enhanced functionalities in microfluidics and microfluidics-enhanced plasmonic devices. The former focuses on plasmonic manipulations of fluids, bubbles, particles, biological cells, and molecules at the micro-/nano-scale. The latter includes technological advances that apply microfluidic principles to enable reconfigurable plasmonic devices and performance-enhanced plasmonic sensors. We conclude with our perspectives on the upcoming challenges, opportunities, and the possible future directions of the emerging field of plasmofluidics. PMID:26140612

Plasmofluidics: Merging Light and Fluids at the Micro-/Nanoscale.

PubMed

Wang, Mingsong; Zhao, Chenglong; Miao, Xiaoyu; Zhao, Yanhui; Rufo, Joseph; Liu, Yan Jun; Huang, Tony Jun; Zheng, Yuebing

2015-09-16

Plasmofluidics is the synergistic integration of plasmonics and micro/nanofluidics in devices and applications in order to enhance performance. There has been significant progress in the emerging field of plasmofluidics in recent years. By utilizing the capability of plasmonics to manipulate light at the nanoscale, combined with the unique optical properties of fluids and precise manipulation via micro/nanofluidics, plasmofluidic technologies enable innovations in lab-on-a-chip systems, reconfigurable photonic devices, optical sensing, imaging, and spectroscopy. In this review article, the most recent advances in plasmofluidics are examined and categorized into plasmon-enhanced functionalities in microfluidics and microfluidics-enhanced plasmonic devices. The former focuses on plasmonic manipulations of fluids, bubbles, particles, biological cells, and molecules at the micro/nanoscale. The latter includes technological advances that apply microfluidic principles to enable reconfigurable plasmonic devices and performance-enhanced plasmonic sensors. The article is concluded with perspectives on the upcoming challenges, opportunities, and possible future directions of the emerging field of plasmofluidics. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel insights of microRNAs in the development of systemic lupus erythematosus.

PubMed

Le, Xiong; Yu, Xiang; Shen, Nan

2017-09-01

To provide a brief overview of recent progress in microRNA biogenesis and homeostasis, its function in immune system and systemic lupus erythematosus (SLE), as well as successful microRNA-based therapy in vivo. Stepwise microRNA biogenesis is elaborately regulated at multiple levels, ranging from transcription to ultimate function. Mature microRNAs have inhibitory effects on various biological molecules, which are crucial for stabilizing and normalizing differentiation and function of immune cells. Abnormality in microRNA expression contributes to dysfunction of lupus immune cells and resident cells in local tissues. Manipulation of dysregulated microRNAs in vivo through microRNA delivery or targeting microRNA might be promising for SLE treatment. Recent advances highlight that microRNAs are important in immunity, lupus autoimmunity and as potential therapy target for SLE.

MicroPET/CT Imaging of an Orthotopic Model of Human Glioblastoma Multiforme and Evaluation of Pulsed Low-Dose Irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Sean S.; Chunta, John L.; Robertson, John M.

2011-07-01

Purpose: Glioblastoma multiforme (GBM) is an aggressive tumor that typically causes death due to local progression. To assess a novel low-dose radiotherapy regimen for treating GBM, we developed an orthotopic murine model of human GBM and evaluated in vivo treatment efficacy using micro-positron-emission tomography/computed tomography (microPET/CT) tumor imaging. Methods: Orthotopic GBM xenografts were established in nude mice and treated with standard 2-Gy fractionation or 10 0.2-Gy pulses with 3-min interpulse intervals, for 7 consecutive days, for a total dose of 14 Gy. Tumor growth was quantified weekly using the Flex Triumph (GE Healthcare/Gamma Medica-Ideas, Waukesha, WI) combined PET-single-photon emission CTmore » (SPECT)-CT imaging system and necropsy histopathology. Normal tissue damage was assessed by counting dead neural cells in tissue sections from irradiated fields. Results: Tumor engraftment efficiency for U87MG cells was 86%. Implanting 0.5 x 10{sup 6} cells produced a 50- to 70-mm{sup 3} tumor in 10 to 14 days. A significant correlation was seen between CT-derived tumor volume and histopathology-measured volume (p = 0.018). The low-dose 0.2-Gy pulsed regimen produced a significantly longer tumor growth delay than standard 2-Gy fractionation (p = 0.045). Less normal neuronal cell death was observed after the pulsed delivery method (p = 0.004). Conclusion: This study successfully demonstrated the feasibility of in vivo brain tumor imaging and longitudinal assessment of tumor growth and treatment response with microPET/CT. Pulsed radiation treatment was more efficacious than the standard fractionated treatment and was associated with less normal tissue damage.« less

MicroPET/CT imaging of an orthotopic model of human glioblastoma multiforme and evaluation of pulsed low-dose irradiation.

PubMed

Park, Sean S; Chunta, John L; Robertson, John M; Martinez, Alvaro A; Oliver Wong, Ching-Yee; Amin, Mitual; Wilson, George D; Marples, Brian

2011-07-01

Glioblastoma multiforme (GBM) is an aggressive tumor that typically causes death due to local progression. To assess a novel low-dose radiotherapy regimen for treating GBM, we developed an orthotopic murine model of human GBM and evaluated in vivo treatment efficacy using micro-positron-emission tomography/computed tomography (microPET/CT) tumor imaging. Orthotopic GBM xenografts were established in nude mice and treated with standard 2-Gy fractionation or 10 0.2-Gy pulses with 3-min interpulse intervals, for 7 consecutive days, for a total dose of 14 Gy. Tumor growth was quantified weekly using the Flex Triumph (GE Healthcare/Gamma Medica-Ideas, Waukesha, WI) combined PET-single-photon emission CT (SPECT)-CT imaging system and necropsy histopathology. Normal tissue damage was assessed by counting dead neural cells in tissue sections from irradiated fields. Tumor engraftment efficiency for U87MG cells was 86%. Implanting 0.5 × 10(6) cells produced a 50- to 70-mm(3) tumor in 10 to 14 days. A significant correlation was seen between CT-derived tumor volume and histopathology-measured volume (p = 0.018). The low-dose 0.2-Gy pulsed regimen produced a significantly longer tumor growth delay than standard 2-Gy fractionation (p = 0.045). Less normal neuronal cell death was observed after the pulsed delivery method (p = 0.004). This study successfully demonstrated the feasibility of in vivo brain tumor imaging and longitudinal assessment of tumor growth and treatment response with microPET/CT. Pulsed radiation treatment was more efficacious than the standard fractionated treatment and was associated with less normal tissue damage. Copyright © 2011 Elsevier Inc. All rights reserved.

A New Micro-holder Device for Local Drug Delivery during In Vivo Whole-cell Recordings.

PubMed

Sáez, María; Ketzef, Maya; Alegre-Cortés, Javier; Reig, Ramón; Silberberg, Gilad

2018-06-15

Focal administration of pharmacological agents during in vivo recordings is a useful technique to study the functional properties of neural microcircuits. However, the lack of visual control makes this task difficult and inaccurate, especially when targeting small and deep regions where spillover to neighboring regions is likely to occur. An additional problem with recording stability arises when combining focal drug administration with in vivo intracellular recordings, which are highly sensitive to mechanical vibrations. To address these technical issues, we designed a micro-holder that enables accurate local application of pharmacological agents during in vivo whole-cell recordings. The holder couples the recording and drug delivery pipettes with adjustable distance between the respective tips adapted to the experimental needs. To test the efficacy of the micro-holder we first performed whole-cell recordings in mouse primary somatosensory cortex (S1) with simultaneous extracellular recordings in S1 and motor cortex (M1), before and after local application of bicuculline methiodide (BMI 200 µM). The blockade of synaptic inhibition resulted in increased amplitudes and rising slopes of "Up states", and shortening of their duration. We then checked the usability of the micro-holder in a deeper brain structure, the striatum. We applied tetrodotoxin (TTX 10 µM) during whole-cell recordings in the striatum, while simultaneously obtaining extracellular recordings in S1 and M1. The focal application of TTX in the striatum blocked Up states in the recorded striatal neurons, without affecting the cortical activity. We also describe two different approaches for precisely releasing the drugs without unwanted leakage along the pipette approach trajectory. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

miR-7 Increases Cisplatin Sensitivity of Gastric Cancer Cells Through Suppressing mTOR

PubMed Central

Lian, Yan-Jun; Dai, Xiang; Wang, Yuan-Jie

2017-01-01

MicroRNAs have been reported to play an important role in diverse biological processes and cancer progression. MicroRNA-7 has been observed to be downregulated in human gastric cancer tissues, but the function of microRNA-7 in gastric cancer has not been well investigated. In this study, we demonstrate that the expression of microRNA-7 was significantly downregulated in 30 pairs of human gastric cancer tissues compared to adjacent normal tissues. Enforced expression of microRNA-7 inhibited cell proliferation and migration abilities of gastric cancer cells, BGC823 and SGC7901. Furthermore, microRNA-7 targeted mTOR in gastric cancer cells. In human clinical specimens, mTOR was higher expressed in gastric cancer tissues compared with adjacent normal tissues. More interestingly, microRNA-7 also sensitizes gastric cancer cells to cisplatin (CDDP) by targeting mTOR. Collectively, our results demonstrate that microRNA-7 is a tumor suppressor microRNA and indicate its potential application for the treatment of human gastric cancer in future. PMID:28693382

Micro Solar Cells with Concentration and Light Trapping Optics

NASA Astrophysics Data System (ADS)

Li, Lanfang; Breuckner, Eric; Corcoran, Christopher; Yao, Yuan; Xu, Lu; Nuzzo, Ralph

2013-03-01

Compared with conventional bulk plate semiconductor solar cells, micro solar cells provide opportunity for novel design geometry and provide test bed for light trapping at the device level as well as module level. Surface recombination, however, will have to be addressed properly as the much increased surface area due to the reduced dimension is more prominent in these devices than conventional solar cells. In this poster, we present experimental demonstration of silicon micro solar cells with concentration and light trapping optics. Silicon micro solar cell with optimized surface passivation and doping profile that exhibit high efficiency is demonstrated. Effective incorporation of high quantum yield fluorescent centers in the polymer matrix into which micro solar cell was encapsulated was investigated for luminescent solar concentration application. Micro-cell on a semi-transparent, nanopatterned reflector formed by soft-imprint lithography was investigated for near field effect related solar conversion performance enhancement. This work is supported by the DOE `Light-Material Interactions in Energy Conversion' Energy Frontier Research Center under grant DE-SC0001293

MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans

PubMed Central

Virant-Klun, Irma; Ståhlberg, Anders; Kubista, Mikael; Skutella, Thomas

2016-01-01

MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in cell-to-cell communication. MicroRNAs, together with exosomes, have a great potential to be used for prognosis, therapy, and biomarkers of different diseases including infertility. The aim of this review paper is to summarize the existent knowledge on microRNAs related to female fertility and cancer: from primordial germ cells and ovarian function, germinal stem cells, oocytes, and embryos to embryonic stem cells. PMID:26664407

Analysis of long-time operation of micro-cogeneration unit with fuel cell

NASA Astrophysics Data System (ADS)

Patsch, Marek; Čaja, Alexander

2015-05-01

Micro-cogeneration is cogeneration with small performance, with maximal electric power up to 50 kWe. On the present, there are available small micro-cogeneration units with small electric performance, about 1 kWe, which are usable also in single family houses or flats. These micro-cogeneration units operate on principle of conventional combustion engine, Stirling engine, steam engine or fuel cell. Micro-cogeneration units with fuel cells are new progressive developing type of units for single family houses. Fuel cell is electrochemical device which by oxidation-reduction reaction turn directly chemical energy of fuel to electric power, secondary products are pure water and thermal energy. The aim of paper is measuring and evaluation of operation parameters of micro-cogeneration unit with fuel cell which uses natural gas as a fuel.

The role of the micro-pattern and nano-topography of hydroxyapatite bioceramics on stimulating osteogenic differentiation of mesenchymal stem cells.

PubMed

Zhao, Cancan; Wang, Xiaoya; Gao, Long; Jing, Linguo; Zhou, Quan; Chang, Jiang

2018-06-01

The micro/nano hybrid structure is considered to be a biomaterial characteristic to stimulate osteogenesis by mimicking the three-dimensional structure of the bone matrix. However, the mechanism of the hybrid structure induced osteogenic differentiation of stem cells is still unknown. For elucidating the mechanisms, one of the challenge is to directly fabricate micro/nano hybrid structure on bioceramics because of its brittleness. In this study, hydroxyapatite (HA) bioceramics with the micro/nano hybrid structure were firstly fabricated via a hydrothermal treatment and template method, and the effect of the different surface structures on the expression of integrins, BMP2 signaling pathways and cell-cell communication was investigated. Interestingly, the results suggested that the osteogenic differentiation induced by micro/nano structures was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, while activated BMP2 could in turn activate integrins and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect. The micro/nano hybrid structure has been found to have synergistic bioactivity on osteogenesis. However, it is still a challenge to fabricate the hybrid structure directly on the bioceramics, and the role of micro- and nano-structure, in particular the mechanism of the micro/nano-hybrid structure induced stem cell differentiation is still unknown. In this study, we firstly fabricated hydroxyapatite bioceramics with the micro/nano hybrid structure, and then investigated the effect of different surface structure on expression of integrins, BMP2 signaling pathways and cell-cell communication. Interestingly, we found that the osteogenic differentiation induced by structure was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, and activated BMP2 could in turn activate some integrin subunits and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Evaluation of the combined use of metronomic zoledronic acid and Coriolus versicolor in intratibial breast cancer mouse model.

PubMed

Ko, Chun-Hay; Yue, Grace Gar-Lee; Gao, Si; Luo, Ke-Wang; Siu, Wing-Sum; Shum, Wai-Ting; Shiu, Hoi-Ting; Lee, Julia Kin-Ming; Li, Gang; Leung, Ping-Chung; Evdokiou, Andreas; Lau, Clara Bik-San

2017-05-23

Coriolus versicolor (CV) is a mushroom traditionally used for strengthening the immune system and nowadays used as immunomodulatory adjuvant in anticancer therapy. Breast cancer usually metastasizes to the skeleton, interrupts the normal bone remodeling process and causes osteolytic bone lesions. The aims of the present study were to evaluate its herb-drug interaction with metronomic zoledronate in preventing cancer propagation, metastasis and bone destruction. Mice inoculated with human breast cancer cells tagged with a luciferase (MDA-MB-231-TXSA) in tibia were treated with CV aqueous extract, mZOL, or the combination of both for 4 weeks. Alteration of the luciferase signals in tibia, liver and lung were quantified using the IVIS imaging system. The skeletal response was evaluated using micro-computed tomography (micro-CT). In vitro experiments were carried out to confirm the in vivo findings. Results showed that combination of CV and mZOL diminished tumor growth without increasing the incidence of lung and liver metastasis in intratibial breast tumor model. The combination therapy also reserved the integrity of bones. In vitro studies demonstrated that combined use of CV and mZOL inhibited cancer cell proliferation and osteoclastogenesis. These findings suggested that combination treatment of CV and mZOL attenuated breast tumor propagation, protected against osteolytic bone lesion without significant metastases. This study provides scientific evidences on the beneficial outcome of using CV together with mZOL in the management of breast cancer and metastasis, which may lead to the development of CV as adjuvant health supplement for the control of breast cancer. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

An Analysis of microRNA Expression in the Myelodysplastic Syndromes Using Hematopoietic Stem Cells

DTIC Science & Technology

2015-10-01

AWARD NUMBER: W81XWH-13-1-0082 TITLE: An Analysis of microRNA Expression in the Myelodysplastic Syndromes Using Hematopoietic Stem Cells ...syndromes (MDS) to identify microRNAs (miRNAs) dysregulated in MDS hematopoietic stem cells (MDS HSCs) as compared with normal HSCs. MiRNAs differentially...the age-related predisposition for the development of MDS. 15. SUBJECT TERMS MicroRNAs, the myelodysplastic syndromes, hematopoietic stem cells

An Analysis of MicroRNA Expression in the Myelodysplastic Syndromes Using Hematopoietic Stem Cells

DTIC Science & Technology

2015-10-01

AWARD NUMBER: W81XWH-13-1-0082 TITLE: An Analysis of microRNA Expression in the Myelodysplastic Syndromes Using Hematopoietic Stem Cells ...syndromes (MDS) to identify microRNAs (miRNAs) dysregulated in MDS hematopoietic stem cells (MDS HSCs) as compared with normal HSCs. MiRNAs differentially...the age-related predisposition for the development of MDS. 15. SUBJECT TERMS MicroRNAs, the myelodysplastic syndromes, hematopoietic stem cells

N-acetylcysteine and endothelial cell injury by sulfur mustard.

PubMed

Atkins, K B; Lodhi, I J; Hurley, L L; Hinshaw, D B

2000-12-01

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells (Toxicol. Appl. Pharmacol. 1996; 141: 568-583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N-acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFkappaB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFkappaB following exposure to HD. The cells were treated with 0-10 mM GSH for 1 h prior to and during exposure to 0 or 500 microM HD for 5-6 h. Cells were also treated with 50 mM NAC or 200 microM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 microM HD for 5-6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFkappaB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 microM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFkappaB binding to its consensus sequence induced by 500 microM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFkappaB, although HD-induced activation of NFkappaB was partially suppressed by NAC at 5 h. Factor NFkappaB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFkappaB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFkappaB activity. The NFkappaB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines.

Cytotoxic activity of quassinoids from Eurycoma longifolia.

PubMed

Miyake, Katsunori; Li, Feng; Tezuka, Yasuhiro; Awale, Suresh; Kadota, Shigetoshi

2010-07-01

Twenty-four quassinoids isolated from Eurycoma longifolia Jack were investigated for their cytotoxicity against a panel of four different cancer cell lines, which includes three murine cell lines [colon 26-L5 carcinoma (colon 26-L5), B16-BL6 melanoma (B16-BL6), Lewis lung carcinoma (LLC)] and a human lung A549 adenocarcinoma (A549) cell line. Among the tested compounds, eurycomalactone (9) displayed the most potent activity against all the tested cell lines; colon 26-L5 (IC50 = 0.70 microM), B16-BL6 (IC50 = 0.59 microM), LLC (IC50 = 0.78 microM), and A549 (IC50 = 0.73 microM). These activities were comparable to clinically used anticancer agent doxorubicin (colon 26-L5, IC50 = 0.76 microM; B16-BL6, IC50 = 0.86 microM; LLC, IC50 = 0.80 microM; A549, IC50 = 0.66 microM).

DynaMiTES - A dynamic cell culture platform for in vitro drug testing PART 1 - Engineering of microfluidic system and technical simulations.

PubMed

Mattern, Kai; Beißner, Nicole; Reichl, Stephan; Dietzel, Andreas

2018-05-01

Conventional safety and efficacy test models, such as animal experiments or static in vitro cell culture models, can often not reliably predict the most promising drug candidates. Therefore, a novel microfluidic cell culture platform, called Dynamic Micro Tissue Engineering System (DynaMiTES), was designed to allow online analysis of drugs permeating through barrier forming tissues under dynamic conditions combined with monitoring of the transepithelial electrical resistance (TEER) by electrodes optimized for homogeneous current distribution. A variety of pre-cultivated cell culture inserts can be integrated and exposed to well controlled dynamic micro flow conditions, resulting in a tightly regulated exposure of the cells to tested drugs, drug formulations and shear forces. With these qualities, the new system can provide more relevant information compared to static measurements. As a first in vitro model, a three-dimensional hemicornea construct consisting of human keratocytes (HCK-Ca) and epithelial cells (HCE-T) was successfully tested in the DynaMiTES. Thereby, we were able to demonstrate the functionality and cell compatibility of this new organ on chip test platform. The modular design of the DynaMiTES allows fast adaptation suitable for the investigation of drug permeation through other important cellular barriers. Copyright © 2017. Published by Elsevier B.V.

Nuclear patterns of human breast cancer cells during apoptosis: characterisation by fractal dimension and co-occurrence matrix statistics.

PubMed

Losa, Gabriele A; Castelli, Christian

2005-11-01

An analytical strategy combining fractal geometry and grey-level co-occurrence matrix (GLCM) statistics was devised to investigate ultrastructural changes in oestrogen-insensitive SK-BR3 human breast cancer cells undergoing apoptosis in vitro. Apoptosis was induced by 1 microM calcimycin (A23187 Ca(2+) ionophore) and assessed by measuring conventional cellular parameters during the culture period. SK-BR3 cells entered the early stage of apoptosis within 24 h of treatment with calcimycin, which induced detectable changes in nuclear components, as documented by increased values of most GLCM parameters and by the general reduction of the fractal dimensions. In these affected cells, morphonuclear traits were accompanied by the reduction of distinct gangliosides and loss of unidentifiable glycolipid molecules at the cell surface. All these changes were shown to be involved in apoptosis before the detection of conventional markers, which were only measurable during the active phases of apoptotic cell death. In overtly apoptotic cells treated with 1 microM calcimycin for 72 h, most nuclear components underwent dramatic ultrastructural changes, including marginalisation and condensation of chromatin, as reflected in a significant reduction of their fractal dimensions. Hence, both fractal and GLCM analyses confirm that the morphological reorganisation of nuclei, attributable to a loss of structural complexity, occurs early in apoptosis.

A Micro-Ark for Cells: Highly Open Porous Polyhydroxyalkanoate Microspheres as Injectable Scaffolds for Tissue Regeneration.

PubMed

Wei, Dai-Xu; Dao, Jin-Wei; Chen, Guo-Qiang

2018-06-19

To avoid large open surgery using scaffold transplants, small-sized cell carriers are employed to repair complexly shaped tissue defects. However, most cell carriers show poor cell adherences and viability. Therefore, polyhydroxyalkanoate (PHA), a natural biopolymer, is used to prepare highly open porous microspheres (OPMs) of 300-360 µm in diameter, combining the advantages of microspheres and scaffolds to serve as injectable carriers harboring proliferating stem cells. In addition to the convenient injection to a defected tissue, and in contrast to poor performances of OPMs made of polylactides (PLA OPMs) and traditional less porous hollow microspheres (PHA HMs), PHA OPMs present suitable surface pores of 10-60 µm and interconnected passages with an average size of 8.8 µm, leading to a high in vitro cell adhesion of 93.4%, continuous proliferation for 10 d and improved differentiation of human bone marrow mesenchymal stem cells (hMSCs). PHA OPMs also support stronger osteoblast-regeneration compared with traditional PHA HMs, PLA OPMs, commercial hyaluronic acid hydrogels, and carrier-free hMSCs in an ectopic bone-formation mouse model. PHA OPMs protect cells against stresses during injection, allowing more living cells to proliferate and migrate to damaged tissues. They function like a micro-Noah's Ark to safely transport cells to a defect tissue. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

NASA Astrophysics Data System (ADS)

Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

2011-11-01

Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

Cytotoxic effects of treosulfan on prostate cancer cell lines.

PubMed

Feyerabend, Susan; Feil, Gerhard; Krug, Jutta; Kassen, Annette; Stenzl, Arnulf

2007-01-01

Despite various therapeutical options in metastatic prostate cancer, the lack of a curative approach motivates further investigations. Treosulfan is an alkylating agent that has proven its indication in the treatment of e.g. ovarian carcinoma. This study focused on the objective of evaluating the effect of in vitro intoxication of human prostate carcinoma cell lines with treosulfan. Human prostate cancer cell lines LNCaP, DU145 and PC3 were treated with treosulfan concentrations from 0.5-500 microM for up to six days. Analysis of cell viability was performed using colorimetric WST-1 assay. Control data were obtained from identical cell lines cultivated without treosulfan. Incubation with treosulfan inhibited cell viability and led to cell death in all cell lines in a dose- and time-dependent manner. After one day, viability of LNCaP, DU145 and PC3 cells was constantly reduced with a dose rate of at least 10 microM (p < 0.001), 10 microM (p < 0.0001) and 100 microM (p < 0.0001) treosulfan, respectively. Minimum dose rates leading to death of nearly all LNCaP, DU145 and PC3 cells were 250 microM, 100 microM and 200 microM treosulfan, respectively. The results demonstrate a sensitivity of prostate carcinoma cells to the cytotoxic activity of treosulfan. Therefore, treosulfan might be a promising compound for novel treatment protocols for prostate cancer.

[Micro fabricated enzyme battery].

PubMed

Sasaki, S; Karube, I

1996-10-01

Although various work has been done in the field of implantable micro actuators such as artificial organs and micro surgery robots, a suitable electric power supply for these is yet to be developed. For this purpose a micro fabricated enzyme fuel cell was developed which uses glucose contained in the human body as a fuel. In order to obtain enough voltage each cell was formed as part of a serial array on a silicon wafer. Glucose solution enters the cells by a capillary effect. In this article fuel cells already developed using biocatalysts are described, and the future possibility of a micro fabricated enzyme battery is discussed.

Self-immolative nanoparticles for simultaneous delivery of microRNA and targeting of polyamine metabolism in combination cancer therapy.

PubMed

Xie, Ying; Murray-Stewart, Tracy; Wang, Yazhe; Yu, Fei; Li, Jing; Marton, Laurence J; Casero, Robert A; Oupický, David

2017-01-28

Combination of anticancer drugs with therapeutic microRNA (miRNA) has emerged as a promising anticancer strategy. However, the promise is hampered by a lack of desirable delivery systems. We report on the development of self-immolative nanoparticles capable of simultaneously delivering miR-34a mimic and targeting dysregulated polyamine metabolism in cancer. The nanoparticles were prepared from a biodegradable polycationic prodrug, named DSS-BEN, which was synthesized from a polyamine analog N 1 ,N 11 -bisethylnorspermine (BENSpm). The nanoparticles were selectively disassembled in the cytoplasm where they released miRNA. Glutathione (GSH)-induced degradation of self-immolative linkers released BENSpm from the DSS-BEN polymers. MiR-34a mimic was effectively delivered to cancer cells as evidenced by upregulation of intracellular miR-34a and downregulation of Bcl-2 as one of the downstream targets of miR-34a. Intracellular BENSpm generated from the degraded nanoparticles induced the expression of rate-limiting enzymes in polyamine catabolism (SMOX, SSAT) and depleted cellular natural polyamines. Simultaneous regulation of polyamine metabolism and miR-34a expression by DSS-BEN/miR-34a not only enhanced cancer cell killing in cultured human colon cancer cells, but also improved antitumor activity in vivo. The reported findings validate the self-immolative nanoparticles as delivery vectors of therapeutic miRNA capable of simultaneously targeting dysregulated polyamine metabolism in cancer, thereby providing an elegant and efficient approach to combination nanomedicines. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel mode of action of the calcium antagonist mibefradil (Ro 40-5967): potent immunosuppression by inhibition of T-cell infiltration through allogeneic endothelium.

PubMed Central

Blaheta, R A; Hailer, N P; Brude, N; Wittig, B; Oppermann, E; Leckel, K; Harder, S; Scholz, M; Weber, S; Encke, A; Markus, B H

1998-01-01

Cyclosporin A reduces the mitotic activity of allosensitized lymphocytes, but fails to limit emigration of these cells into the donor organ. However, the modulation of both lymphocyte proliferation and infiltration are desirable characteristics of immunosuppressive therapy. The calcium-channel blocker, verapamil, has recently been shown to effectively prevent the transmigration of CD4+ and CD8+ T cells through allogeneic endothelium. Mibefradil (Ro 40-5967) represents a new generation of calcium antagonists with high potency and long-term activity. To evaluate the immunosuppressive potential of this drug, the influence of mibefradil on lymphocyte adhesion to, horizontal locomotion along, and penetration through allogeneic endothelium (HUVEC) was performed. When lymphocytes were prestimulated for 24 hr with mibefradil, adhesion and penetration were dose-dependently reduced. The adhesion ID50 values were 3.4 microM (CD4+ T cells) versus 9.2 microM (CD8+ T cells) and 2.1 microM (CD4+ T cells) versus 3.9 microM (CD8+ T cells) with regard to penetration. Mibefradil also effectively blocked horizontal locomotion. Specific down-regulation of T-cell binding to the P-selection receptor (ID50: CD4+ T cells, 0.8 microM: CD8+ T cells, 1.2 microM) and to the intracellular adhesion molecule-1 (ICAM-1) receptor (ID50: CD4+ T cells, 1.9 microM; CD8+ T cells, 1.5 microM) by mibefradil seems to be responsible for the decreased adhesion and penetration rates. Reduction of intracellular F-actin in T lymphocytes could diminish cell locomotion. In conclusion, the potent suppressive properties of mibefradil support its use as a co-medication in cyclosporin A-based immunosuppressive therapy. PMID:9741343

Novel mode of action of the calcium antagonist mibefradil (Ro 40-5967): potent immunosuppression by inhibition of T-cell infiltration through allogeneic endothelium.

PubMed

Blaheta, R A; Hailer, N P; Brude, N; Wittig, B; Oppermann, E; Leckel, K; Harder, S; Scholz, M; Weber, S; Encke, A; Markus, B H

1998-06-01

Cyclosporin A reduces the mitotic activity of allosensitized lymphocytes, but fails to limit emigration of these cells into the donor organ. However, the modulation of both lymphocyte proliferation and infiltration are desirable characteristics of immunosuppressive therapy. The calcium-channel blocker, verapamil, has recently been shown to effectively prevent the transmigration of CD4+ and CD8+ T cells through allogeneic endothelium. Mibefradil (Ro 40-5967) represents a new generation of calcium antagonists with high potency and long-term activity. To evaluate the immunosuppressive potential of this drug, the influence of mibefradil on lymphocyte adhesion to, horizontal locomotion along, and penetration through allogeneic endothelium (HUVEC) was performed. When lymphocytes were prestimulated for 24 hr with mibefradil, adhesion and penetration were dose-dependently reduced. The adhesion ID50 values were 3.4 microM (CD4+ T cells) versus 9.2 microM (CD8+ T cells) and 2.1 microM (CD4+ T cells) versus 3.9 microM (CD8+ T cells) with regard to penetration. Mibefradil also effectively blocked horizontal locomotion. Specific down-regulation of T-cell binding to the P-selection receptor (ID50: CD4+ T cells, 0.8 microM: CD8+ T cells, 1.2 microM) and to the intracellular adhesion molecule-1 (ICAM-1) receptor (ID50: CD4+ T cells, 1.9 microM; CD8+ T cells, 1.5 microM) by mibefradil seems to be responsible for the decreased adhesion and penetration rates. Reduction of intracellular F-actin in T lymphocytes could diminish cell locomotion. In conclusion, the potent suppressive properties of mibefradil support its use as a co-medication in cyclosporin A-based immunosuppressive therapy.

Combined optical resolution photoacoustic and fluorescence micro-endoscopy

NASA Astrophysics Data System (ADS)

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J.

2012-02-01

We present a new micro-endoscopy system combining real-time C-scan optical-resolution photoacoustic micro-endoscopy (OR-PAME), and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the OR-PAM sub-system is capable of imaging with a resolution of ~ 7μm. The fluorescence sub-system consists of a diode laser with 445 nm-centered emissions as the light source, an objective lens and a CCD camera. Proflavine, a FDA approved drug for human use, is used as the fluorescent contrast agent by topical application. The fluorescence system does not require any mechanical scanning. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single mode fibers. The absorption of Proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural and functional information given by OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping researchers and clinicians visualize angiogenesis, effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Microfluidic 3D cell culture: potential application for tissue-based bioassays

PubMed Central

Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

2014-01-01

Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

Quantitative, Structural and Image-based Mechanical Analysis of Nonunion Fracture Repaired by Genetically Engineered Mesenchymal Stem Cells

PubMed Central

Kallai, Ilan; van Lenthe, G. Harry; Ruffoni, Davide; Zilberman, Yoram; Müller, Ralph; Pelled, Gadi; Gazit, Dan

2010-01-01

Stem cell-mediated gene therapy for fracture repair, utilizes genetically engineered mesenchymal stem cells (MSCs) for the induction of bone growth and is considered a promising approach in skeletal tissue regeneration. Previous studies have shown that murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). Nanoindentation studies of bone tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine bone, eight weeks post treatment. In the present study we sought to investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius bones, following the implantation of MSCs. High resolution micro computed tomography (Micro-CT) was performed 10 and 35 weeks post MSC implantation, followed by micro finite element (Micro-FE) analysis. The results have shown that the regenerated bone tissue remodels over time, as indicated by a significant decrease in bone volume, total volume and connectivity density combined with an increase in mineral density. In addition, the axial stiffness of limbs repaired with MSCs was 2 to 1.5 times higher compared to the contralateral intact limbs, at 10 and 35 weeks post treatment. These results could be attributed to the fusion that occurred between in the ulna and radius bones. In conclusion, although MSCs induce bone formation, which exceeds the fracture site, significant remodeling of the repair callus occurs over time. In addition, limbs treated with an MSC graft demonstrated superior biomechanical properties, which could indicate the clinical benefit of future MSC application in nonunion fracture repair. PMID:20471652

High-definition micropatterning method for hard, stiff and brittle polymers.

PubMed

Zhao, Yiping; Truckenmuller, Roman; Levers, Marloes; Hua, Wei-Shu; de Boer, Jan; Papenburg, Bernke

2017-02-01

Polystyrene (PS) is the most commonly used material in cell culture devices, such as Petri dishes, culture flasks and well plates. Micropatterning of cell culture substrates can significantly affect cell-material interactions leading to an increasing interest in the fabrication of topographically micro-structured PS surfaces. However, the high stiffness combined with brittleness of PS (elastic modulus 3-3.5GPa) makes high-quality patterning into PS difficult when standard hard molds, e.g. silicon and nickel, are used as templates. A new and robust scheme for easy processing of large-area high-density micro-patterning into PS film is established using nanoimprinting lithography and standard hot embossing techniques. Including an extra step through an intermediate PDMS mold alone does not result in faithful replication of the large area, high-density micropattern into PS. Here, we developed an approach using an additional intermediate mold out of OrmoStamp, which allows for high-quality and large-area micro-patterning into PS. OrmoStamp was originally developed for UV nanoimprint applications; this work demonstrates for the first time that OrmoStamp is a highly adequate material for micro-patterning of PS through hot embossing. Our proposed processing method achieves high-quality replication of micropatterns in PS, incorporating features with high aspect ratio (4:1, height:width), high density, and over a large pattern area. The proposed scheme can easily be adapted for other large-area and high-density micropatterns of PS, as well as other stiff and brittle polymers. Copyright © 2016 Elsevier B.V. All rights reserved.

Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beer, Neil Reginald; Colston, Jr, Billy W.

An apparatus for chip-based sorting, amplification, detection, and identification of a sample having a planar substrate. The planar substrate is divided into cells. The cells are arranged on the planar substrate in rows and columns. Electrodes are located in the cells. A micro-reactor maker produces micro-reactors containing the sample. The micro-reactor maker is positioned to deliver the micro-reactors to the planar substrate. A microprocessor is connected to the electrodes for manipulating the micro-reactors on the planar substrate. A detector is positioned to interrogate the sample contained in the micro-reactors.

Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM.

PubMed

Deegan, Anthony J; Cinque, Gianfelice; Wehbe, Katia; Konduru, Sandeep; Yang, Ying

2015-02-01

One novel tissue engineering approach to mimic in vivo bone formation is the use of aggregate or micromass cultures. Various qualitative and quantitative techniques, such as histochemical staining, protein assay kits and RT-PCR, have been used previously on cellular aggregate studies to investigate how these intricate arrangements lead to mature bone tissue. However, these techniques struggle to reveal spatial and temporal distribution of proliferation and mineralization simultaneously. Synchrotron-based Fourier transform infrared microspectroscopy (micro-FTIR) offers a unique insight at the molecular scale by coupling high IR sensitivity to organic matter with the high spatial resolution allowed by diffraction limited SR microbeam. This study is set to investigate the effects of culture duration and aggregate size on the dynamics and spatial distribution of calcification in engineered bone aggregates by a combination of micro-FTIR and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX). A murine bone cell line has been used, and small/large bone aggregates have been induced using different chemically treated culture substrates. Our findings suggest that bone cell aggregate culturing can greatly increase levels of mineralization over short culture periods. The size of the aggregates influences mineralisation rates with larger aggregates mineralizing at a faster rate than their smaller counterparts. The micro-FTIR mapping has demonstrated that mineralization in the larger aggregates initiated from the periphery and spread to the centre, whilst the smaller aggregates have more minerals in the centre at the early stage and deposited more in the periphery after further culturing, implying that aggregate size influences calcification distribution and development over time. SEM/EDX data correlates well with the micro-FTIR results for the total mineral content. Thus, synchrotron-based micro-FTIR can accurately track mineralization process/mechanism in the engineered bone.

Insulin-releasing action of the novel antidiabetic agent BTS 67 582.

PubMed

McClenaghan, N H; Flatt, P R; Bailey, C J

1998-02-01

1. BTS 67582 (1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate) is a novel antidiabetic agent with a short-acting insulin-releasing effect. This study examined its mode of action in the clonal B-cell line BRIN-BD11. 2. BTS 67582 increased insulin release from BRIN-BD11 cells in a concentration-dependent manner (10[-8] to 10[-4] M) at both non-stimulating (1.1 mM) and stimulating (16.7 mM) concentrations of glucose. 3. BTS 67582 (10[-4] M) potentiated the insulin-releasing effect of a depolarizing concentration of K+ (30 mM), whereas the K+ channel openers pinacidil (400 microM) and diazoxide (300 microM) inhibited BTS 67582-induced release. 4. Suppression of Ca+ channel activity with verapamil (20 microM) reduced the insulin-releasing effect of BTS 67582 (10[-4] M). 5. BTS 67582 (10[-4] M) potentiated insulin release induced by amino acids (10 mM), and enhanced the combined stimulant effects of glucose plus either the fatty acid palmitate (10 mM), or agents which raise intracellular cyclic AMP concentrations (25 microM forskolin and 1 mM isobutylmethylxanthine), or the cholinoceptor agonist carbachol (100 microM). 6. Inhibition of glucose-stimulated insulin release by adrenaline or noradrenaline (10 microM) was partially reversed by BTS 67582 (10[-4] M). 7. These data suggest that the insulin-releasing effect of BTS 67582 involves regulation of ATP-sensitive K+ channel activity and Ca2+ influx, and that the drug augments the stimulant effects of nutrient insulin secretagogues and agents which enhance adenylate cyclase and phospholipase C. BTS 67582 may also exert insulin-releasing effects independently of ATP-sensitive K+ channel activity.

Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host.

PubMed

Tsetsarkin, Konstantin A; Liu, Guangping; Kenney, Heather; Hermance, Meghan; Thangamani, Saravanan; Pletnev, Alexander G

2016-09-13

Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates.

MG132 as a proteasome inhibitor induces cell growth inhibition and cell death in A549 lung cancer cells via influencing reactive oxygen species and GSH level.

PubMed

Han, Yong Hwan; Park, Woo Hyun

2010-07-01

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In the present study, we evaluated the effects of MG132 on the growth of A549 lung cancer cells in relation to cell growth, ROS and glutathione (GSH) levels. Treatment with MG132 inhibited the growth of A549 cells with an IC(50) of approximately 20 microM at 24 hours. DNA flow cytometric analysis indicated that 0.5 approximately 30 microM MG132 induced a G1 phase arrest of the cell cycle in A549 cells. Treatment with 10 or 30 microM MG132 also induced apoptosis, as evidenced by sub-G1 cells and annexin V staining cells. This was accompanied by the loss of mitochondrial membrane potential (MMP; Delta psi m). The intracellular ROS levels including O(2) (*-) were strongly increased in 10 or 30 microM MG132-treated A549 cells but were down-regulated in 0.1, 0.5 or 1 microM MG132-treated cells. Furthermore, 10 or 30 microM MG132 increased mitochondrial O(2) (*- ) level but 0.1, 0.5 or 1 microM MG132 decreased that. In addition, 10 or 30 microM MG132 induced GSH depletion in A549 cells. In conclusion, MG132 inhibited the growth of human A549 cells via inducing the cell cycle arrest as well as triggering apoptosis, which was in part correlated with the changes of ROS and GSH levels. Our present data provide important information on the anti-growth mechanisms of MG132 in A549 lung cancer cells in relation to ROS and GSH.

A three-dimensional computational fluid dynamics model of shear stress distribution during neotissue growth in a perfusion bioreactor.

PubMed

Guyot, Y; Luyten, F P; Schrooten, J; Papantoniou, I; Geris, L

2015-12-01

Bone tissue engineering strategies use flow through perfusion bioreactors to apply mechanical stimuli to cells seeded on porous scaffolds. Cells grow on the scaffold surface but also by bridging the scaffold pores leading a fully filled scaffold following the scaffold's geometric characteristics. Current computational fluid dynamic approaches for tissue engineering bioreactor systems have been mostly carried out for empty scaffolds. The effect of 3D cell growth and extracellular matrix formation (termed in this study as neotissue growth), on its surrounding fluid flow field is a challenge yet to be tackled. In this work a combined approach was followed linking curvature driven cell growth to fluid dynamics modeling. The level-set method (LSM) was employed to capture neotissue growth driven by curvature, while the Stokes and Darcy equations, combined in the Brinkman equation, provided information regarding the distribution of the shear stress profile at the neotissue/medium interface and within the neotissue itself during growth. The neotissue was assumed to be micro-porous allowing flow through its structure while at the same time allowing the simulation of complete scaffold filling without numerical convergence issues. The results show a significant difference in the amplitude of shear stress for cells located within the micro-porous neo-tissue or at the neotissue/medium interface, demonstrating the importance of taking along the neotissue in the calculation of the mechanical stimulation of cells during culture.The presented computational framework is used on different scaffold pore geometries demonstrating its potential to be used a design as tool for scaffold architecture taking into account the growing neotissue. Biotechnol. Bioeng. 2015;112: 2591-2600. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

A three dimensional scaffold with precise micro-architecture and surface micro-textures

PubMed Central

Mata, Alvaro; Kim, Eun Jung; Boehm, Cynthia A.; Fleischman, Aaron J.; Muschler, George F.; Roy, Shuvo

2013-01-01

A three-dimensional (3D) structure comprising precisely defined microarchitecture and surface micro-textures, designed to present specific physical cues to cells and tissues, may provide an efficient scaffold in a variety of tissue engineering and regenerative medicine applications. We report a fabrication technique based on microfabrication and soft lithography that permits for the development of 3D scaffolds with both precisely engineered architecture and tailored surface topography. The scaffold fabrication technique consists of three key steps starting with microfabrication of a mold using an epoxy-based photoresist (SU-8), followed by dual-sided molding of a single layer of polydimethylsiloxane (PDMS) using a mechanical jig for precise motion control; and finally, alignment, stacking, and adhesion of multiple PDMS layers to achieve a 3D structure. This technique was used to produce 3D Texture and 3D Smooth PDMS scaffolds, where the surface topography comprised 10 μm-diameter/height posts and smooth surfaces, respectively. The potential utility of the 3D microfabricated scaffolds, and the role of surface topography, were subsequently investigated in vitro with a combined heterogeneous population of adult human stem cells and their resultant progenitor cells, collectively termed connective tissue progenitors (CTPs), under conditions promoting the osteoblastic phenotype. Examination of bone-marrow derived CTPs cultured on the 3D Texture scaffold for 9 days revealed cell growth in three dimensions and increased cell numbers compared to those on the 3D Smooth scaffold. Furthermore, expression of alkaline phosphatase mRNA was higher on the 3D Texture scaffold, while osteocalcin mRNA expression was comparable for both types of scaffolds. PMID:19524292

The effect of Taurolidine on adherent and floating subpopulations of melanoma cells.

PubMed

Shrayer, D P; Lukoff, H; King, T; Calabresi, P

2003-04-01

The annual incidence of malignant melanoma is estimated at 10-12 per 100000 inhabitants in countries of Central Europe and the US, with more recent estimates showing a dramatic upward trend. Taurolidine (Carter/Wallace, Cranberry, NJ) is a novel, potentially effective, antitumor chemotherapeutic agent. We hypothesized that Taurolidine could inhibit the growth, induce apoptosis, affect the cell cycle and change morphology of melanoma cells. We expected this process to be different in adherent and floating subpopulations that may be reflective of solid tumors and their metastases. Analysis of MNT-1 human and B16F10 murine melanoma cells showed that at 72 h the IC(50) of Taurolidine was 25.4+/-3.3 microM for MNT-1 human melanoma cells and 30.9+/-3.6 microM for B16F10 murine melanoma cells. Taurolidine induced DNA fragmentation of melanoma cells in a dose-dependent manner. Taurolidine (75 and 100 microM) induced 52-97% Annexin-V binding (apoptosis), respectively. Evaluation of cell cycle after 72 h exposure to Taurolidine (0-100 microM) revealed that the percentage of melanoma cells in S phase increased from 27 to 40% in the adherent subpopulation and from 33 to 49% in the floating subpopulation. Phase contrast microscopy revealed a marked swelling of melanoma cells and decreasing cell numbers in adherent subpopulation starting at 24 h with 25 microM Taurolidine. Shrinkage of cells dominated at 75-100 microM Taurolidine. Using Cytospin assay in the floating population, we observed swelling of melanoma cells induced by 25-100 micro Taurolidine and appearance of giant (multinuclear) forms resulting from exposure to 75-100 micro Taurolidine. Some floating cells with normal morphology were observed with low concentrations of Taurolidine (0-25 microM). These data show that effects of Taurolidine may be different in adherent and floating subpopulations of melanoma cells. More importantly, floating subpopulations that may contain some viable melanoma cells, may be reflective of potential metastasis after treatment of solid tumors in vivo.

Characterization of slowly inactivating KV{alpha} current in rabbit pulmonary neuroepithelial bodies: effects of hypoxia and nicotine.

PubMed

Fu, Xiao Wen; Nurse, Colin; Cutz, Ernest

2007-10-01

Pulmonary neuroepithelial bodies (NEB) form innervated cell clusters that express voltage-activated currents and function as airway O(2) sensors. We investigated A-type K(+) currents in NEB cells using neonatal rabbit lung slice preparation. The whole cell K(+) current was slowly inactivating with activation threshold of approximately -30 mV. This current was blocked approximately 27% by blood-depressing substance I (BDS-I; 3 microM), a selective blocker of Kv3.4 subunit, and reduced approximately 20% by tetraethylammonium (TEA; 100 microM). The BDS-I-sensitive component had an average peak value of 189 +/- 14 pA and showed fast inactivation kinetics that could be fitted by one-component exponential function with a time constant of (tau1) 77 +/- 10 ms. This Kv slowly inactivating current was also blocked by heteropodatoxin-2 (HpTx-2; 0.2 microM), a blocker of Kv4 subunit. The HpTx-2-sensitive current had an average peak value of 234 +/- 23 pA with a time constant (tau) 82 +/- 11 ms. Hypoxia (Po(2) = 15-20 mmHg) inhibited the slowly inactivating K(+) current by approximately 47%, during voltage steps from -30 to +30 mV, and no further inhibition occurred when TEA was combined with hypoxia. Nicotine at concentrations of 50 and 100 microM suppressed the slowly inactivating K(+) current by approximately 24 and approximately 40%, respectively. This suppression was not reversed by mecamylamine suggesting a direct effect of nicotine on these K(+) channels. In situ hybridization experiments detected expression of mRNAs for Kv3.4 and Kv4.3 subunits, while double-label immunofluorescence confirmed membrane localization of respective channel proteins in NEB cells. These studies suggest that the hypoxia-sensitive current in NEB cells is carried by slowly inactivating A-type K(+) channels, which underlie their oxygen-sensitive potassium currents, and that exposure to nicotine may directly affect their function, contributing to smoking-related lung disease.

Application of advanced cytometric and molecular technologies to minimal residual disease monitoring

NASA Astrophysics Data System (ADS)

Leary, James F.; He, Feng; Reece, Lisa M.

2000-04-01

Minimal residual disease monitoring presents a number of theoretical and practical challenges. Recently it has been possible to meet some of these challenges by combining a number of new advanced biotechnologies. To monitor the number of residual tumor cells requires complex cocktails of molecular probes that collectively provide sensitivities of detection on the order of one residual tumor cell per million total cells. Ultra-high-speed, multi parameter flow cytometry is capable of analyzing cells at rates in excess of 100,000 cells/sec. Residual tumor selection marker cocktails can be optimized by use of receiver operating characteristic analysis. New data minimizing techniques when combined with multi variate statistical or neural network classifications of tumor cells can more accurately predict residual tumor cell frequencies. The combination of these techniques can, under at least some circumstances, detect frequencies of tumor cells as low as one cell in a million with an accuracy of over 98 percent correct classification. Detection of mutations in tumor suppressor genes requires insolation of these rare tumor cells and single-cell DNA sequencing. Rare residual tumor cells can be isolated at single cell level by high-resolution single-cell cell sorting. Molecular characterization of tumor suppressor gene mutations can be accomplished using a combination of single- cell polymerase chain reaction amplification of specific gene sequences followed by TA cloning techniques and DNA sequencing. Mutations as small as a single base pair in a tumor suppressor gene of a single sorted tumor cell have been detected using these methods. Using new amplification procedures and DNA micro arrays it should be possible to extend the capabilities shown in this paper to screening of multiple DNA mutations in tumor suppressor and other genes on small numbers of sorted metastatic tumor cells.

Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits.

PubMed

Wang, Zhifa; Hu, Hanqing; Li, Zhijin; Weng, Yanming; Dai, Taiqiang; Zong, Chunlin; Liu, Yanpu; Liu, Bin

2016-04-01

Techniques that use sheets of cells have been successfully used in various types of tissue regeneration, and platelet-rich fibrin (PRF) can be used as a source of growth factors to promote angiogenesis. We have investigated the effects of the combination of PRF and sheets of mesenchymal stem cells (MSC) from bone marrow on the restoration of bone in critical-size calvarial defects in rabbits to find out whether the combination promotes bony healing. Sheets of MSC and PRF were prepared from the same donor. We then implanted the combined MSC and PRF in critical-size calvarial defects in rabbits and assessed bony restoration by microcomputed tomography (microCT) and histological analysis. The results showed that PRF significantly increased bony regeneration at 8 weeks after implantation of sheets of MSC and PRF compared with sheets of MSC alone (p=0.0048). Our results indicate that the combination of sheets of MSC and PRF increases bone regeneration in critical-size calvarial defects in rabbits, and provides a new way to improve skeletal healing. Copyright © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Mission Applicability Assessment of Integrated Power Components and Systems

NASA Technical Reports Server (NTRS)

Raffaelle, R. P.; Hepp, A. F.; Landis, G. A.; Hoffman, D. J.

2002-01-01

The need for smaller lightweight autonomous power systems has recently increased with the increasing focus on micro- and nanosatellites. Small area high-efficiency thin film batteries and solar cells are an attractive choice for such applications. The NASA Glenn Research Center, Johns Hopkins Applied Physics Laboratory, Lithium Power Technologies, MicroSat Systems, and others, have been working on the development of autonomous monolithic packages combining these elements or what are called integrated power supplies (IPS). These supplies can be combined with individual satellite components and are capable of providing continuous power even under intermittent illumination associated with a spinning or Earth orbiting satellite. This paper discusses the space mission applicability, benefits, and current development efforts associated with integrated power supply components and systems. The characteristics and several mission concepts for an IPS that combines thin-film photovoltaic power generation with thin-film lithium ion energy storage are described. Based on this preliminary assessment, it is concluded that the most likely and beneficial application of an IPS will be for small "nanosatellites" or in specialized applications serving as a decentralized or as a distributed power source or uninterruptible power supply.

Nano scaffolds and stem cell therapy in liver tissue engineering

NASA Astrophysics Data System (ADS)

Montaser, Laila M.; Fawzy, Sherin M.

2015-08-01

Tissue engineering and regenerative medicine have been constantly developing of late due to the major progress in cell and organ transplantation, as well as advances in materials science and engineering. Although stem cells hold great potential for the treatment of many injuries and degenerative diseases, several obstacles must be overcome before their therapeutic application can be realized. These include the development of advanced techniques to understand and control functions of micro environmental signals and novel methods to track and guide transplanted stem cells. A major complication encountered with stem cell therapies has been the failure of injected cells to engraft to target tissues. The application of nanotechnology to stem cell biology would be able to address those challenges. Combinations of stem cell therapy and nanotechnology in tissue engineering and regenerative medicine have achieved significant advances. These combinations allow nanotechnology to engineer scaffolds with various features to control stem cell fate decisions. Fabrication of Nano fiber cell scaffolds onto which stem cells can adhere and spread, forming a niche-like microenvironment which can guide stem cells to proceed to heal damaged tissues. In this paper, current and emergent approach based on stem cells in the field of liver tissue engineering is presented for specific application. The combination of stem cells and tissue engineering opens new perspectives in tissue regeneration for stem cell therapy because of the potential to control stem cell behavior with the physical and chemical characteristics of the engineered scaffold environment.

Simulation of micro/nano electroporation for cell transfection

NASA Astrophysics Data System (ADS)

Zhang, Guocheng; Fan, Na; Jiang, Hai; Guo, Jian; Peng, Bei

2018-03-01

The 3D micro/nano electroporation for transfection has become a powerful biological cell research technique with the development of micro-nano manufacturing technology. The micro channels connected the cells with transfection reagents on the chip were important to the transmemnbrane potentical, which directly influences the electroporation efficiency. In this study, a two-dimensional model for electroporation of cells was designed to address the effects of channels’ sizes and number on transmembrane potential. The simulation results indicated that the transmembrane potential increased with increasing size of channels’ entrances. Moreover, compared with single channel entrance, the transmembrane potential was higher when the cells located at multiple channels entrances. These results suggest that it IS required to develop higher micro manufacturing technology to create channels as we expected size.

Synchrotron X-ray micro-tomography at the Advanced Light Source: Developments in high-temperature in-situ mechanical testing

NASA Astrophysics Data System (ADS)

Barnard, Harold S.; MacDowell, A. A.; Parkinson, D. Y.; Mandal, P.; Czabaj, M.; Gao, Y.; Maillet, E.; Blank, B.; Larson, N. M.; Ritchie, R. O.; Gludovatz, B.; Acevedo, C.; Liu, D.

2017-06-01

At the Advanced Light Source (ALS), Beamline 8.3.2 performs hard X-ray micro-tomography under conditions of high temperature, pressure, mechanical loading, and other realistic conditions using environmental test cells. With scan times of 10s-100s of seconds, the microstructural evolution of materials can be directly observed over multiple time steps spanning prescribed changes in the sample environment. This capability enables in-situ quasi-static mechanical testing of materials. We present an overview of our in-situ mechanical testing capabilities and recent hardware developments that enable flexural testing at high temperature and in combination with acoustic emission analysis.

microRNAs as mediators and communicators between cancer cells and the tumor micro-environment

PubMed Central

Kohlhapp, Frederick J.; Mitra, Anirban K.; Lengyel, Ernst; Peter, Marcus E.

2015-01-01

Cancer cells grow in an environment comprised of multiple components that support tumor growth and contribute to therapy resistance. Major cell types in the tumor micro-environment are fibroblasts, endothelial cells and infiltrating immune cells all of which communicate with cancer cells. One way that these cell types promote cancer progression is by altering expression of miRNAs, small noncoding RNAs that negatively regulate protein expression, either in the cancer cells or in associated normal cells. Changes in miRNA expression can be brought about by direct interaction between the stromal cells and cancer cells, by paracrine factors secreted by any of the cell types, or even through direct communication between cells through secreted miRNAs. Understanding the role of miRNAs in the complex interactions between the tumor and cells in its micro-environment is necessary if we are to understand tumor progression and devise new treatments. PMID:25867073

Modulation in vitro and in vivo of cytotoxicity but not cellular levels of doxorubicin by the calmodulin inhibitor trifluoperazine is dependent on the level of resistance.

PubMed Central

Ganapathi, R.; Schmidt, H.; Grabowski, D.; Melia, M.; Ratliff, N.

1988-01-01

The role of the calmodulin inhibitor trifluoperazine (TFP) in modulating the cellular levels and cytotoxicity in vitro and antitumour effects in vivo of doxorubicin (DOX), was evaluated in progressively DOX-resistant (5- to 40-fold) sublines of B16-BL6 mouse melanoma. In parental-sensitive B16-BL6 cells treated for 3 h, the IC50 of DOX was 0.1 microgram ml-1, and a less than 2-fold enhancement in DOX cell kill in the presence of a noncytotoxic concentration of 5 microM TFP was observed. However, in the DOX-resistant sublines, the IC50 was 0.7 to 5.0 micrograms ml-1 DOX in the absence of 5 microM TFP and 0.3 to 0.7 microgram ml-1 DOX in the presence of 5 microM TFP. The 2- to 7.5-fold decrease in the IC50 of DOX in the presence of 5 microM TFP, was dependent on the level of DOX-resistance in the various sublines. Compared to parental-sensitive cells, a 2-fold decrease in DOX-accumulation was evident only in the 40-fold DOX-resistant subline. Further, maximal enhancement (50%) of cellular DOX accumulation in the presence of 5 microM TFP was observed only in the 40-fold resistant cells treated with 5.0 micrograms ml-1 DOX. Retention of DOX in the 40-fold resistant subline was only 20% lower than similarly treated sensitive cells, and the inclusion of TFP increased DOX retention less than 10-15%. Antitumour studies in mice with experimental pulmonary metastases revealed that although DOX and DOX plus TFP had similar antitumour activity with the parental sensitive B16-BL6 cells, the combination of DOX plus TFP was significantly more effective than DOX alone with the DOX-resistant sublines. No overt toxicity was observed in normal mice treated with doses of TFP, DOX or DOX plus TFP used for in vivo chemotherapy studies. Results from this study suggest that gross cellular DOX levels do not appear to correlate with the magnitude of resistance, and the effects of TFP in modulating DOX resistance is possibly due to mechanisms other than mere alterations in cellular drug accumulation and/or retention. PMID:3179186

Fabrication of a Flexible Micro CO Sensor for Micro Reformer Applications

PubMed Central

Lee, Chi-Yuan; Chang, Chi-Chung; Lo, Yi-Man

2010-01-01

Integration of a reformer and a proton exchange membrane fuel cell (PEMFC) is problematic due to the presence in the gas from the reforming process of a slight amount of carbon monoxide. Carbon monoxide poisons the catalyst of the proton exchange membrane fuel cell subsequently degrading the fuel cell performance, and necessitating the sublimation of the reaction gas before supplying to fuel cells. Based on the use of micro-electro-mechanical systems (MEMS) technology to manufacture flexible micro CO sensors, this study elucidates the relation between a micro CO sensor and different SnO2 thin film thicknesses. Experimental results indicate that the sensitivity increases at temperatures ranging from 100–300 °C. Additionally, the best sensitivity is obtained at a specific temperature. For instance, the best sensitivity of SnO2 thin film thickness of 100 nm at 300 °C is 59.3%. Moreover, a flexible micro CO sensor is embedded into a micro reformer to determine the CO concentration in each part of a micro reformer in the future, demonstrating the inner reaction of a micro reformer in depth and immediate detection. PMID:22163494

Optical trapping for analytical biotechnology.

PubMed

Ashok, Praveen C; Dholakia, Kishan

2012-02-01

We describe the exciting advances of using optical trapping in the field of analytical biotechnology. This technique has opened up opportunities to manipulate biological particles at the single cell or even at subcellular levels which has allowed an insight into the physical and chemical mechanisms of many biological processes. The ability of this technique to manipulate microparticles and measure pico-Newton forces has found several applications such as understanding the dynamics of biological macromolecules, cell-cell interactions and the micro-rheology of both cells and fluids. Furthermore we may probe and analyse the biological world when combining trapping with analytical techniques such as Raman spectroscopy and imaging. Copyright © 2011 Elsevier Ltd. All rights reserved.

A prediction of cell differentiation and proliferation within a collagen-glycosaminoglycan scaffold subjected to mechanical strain and perfusive fluid flow.

PubMed

Stops, A J F; Heraty, K B; Browne, M; O'Brien, F J; McHugh, P E

2010-03-03

Mesenchymal stem cell (MSC) differentiation can be influenced by biophysical stimuli imparted by the host scaffold. Yet, causal relationships linking scaffold strain magnitudes and inlet fluid velocities to specific cell responses are thus far underdeveloped. This investigation attempted to simulate cell responses in a collagen-glycosaminoglycan (CG) scaffold within a bioreactor. CG scaffold deformation was simulated using micro-computed tomography (CT) and an in-house finite element solver (FEEBE/linear). Similarly, the internal fluid velocities were simulated using the afore-mentioned microCT dataset with a computational fluid dynamics solver (ANSYS/CFX). From the ensuing cell-level mechanics, albeit octahedral shear strain or fluid velocity, the proliferation and differentiation of the representative cells were predicted from deterministic functions. Cell proliferation patterns concurred with previous experiments. MSC differentiation was dependent on the level of CG scaffold strain and the inlet fluid velocity. Furthermore, MSC differentiation patterns indicated that specific combinations of scaffold strains and inlet fluid flows cause phenotype assemblies dominated by single cell types. Further to typical laboratory procedures, this predictive methodology demonstrated loading-specific differentiation lineages and proliferation patterns. It is hoped these results will enhance in-vitro tissue engineering procedures by providing a platform from which the scaffold loading applications can be tailored to suit the desired tissue. Copyright 2009 Elsevier Ltd. All rights reserved.

A micro-Raman spectroscopic investigation of leukemic U-937 cells in aged cultures

NASA Astrophysics Data System (ADS)

Fazio, Enza; Trusso, Sebastiano; Franco, Domenico; Nicolò, Marco Sebastiano; Allegra, Alessandro; Neri, Fortunato; Musolino, Caterina; Guglielmino, Salvatore P. P.

2016-04-01

Recently it has been shown that micro-Raman spectroscopy combined with multivariate analysis is able to discriminate among different types of tissues and tumoral cells by the detection of significant alterations and/or reorganizations of complex biological molecules, such as nucleic acids, lipids and proteins. Moreover, its use, being in principle a non-invasive technique, appears an interesting clinical tool for the evaluation of the therapeutical effects and of the disease progression. In this work we analyzed molecular changes in aged cultures of leukemia model U937 cells with respect to fresh cultures of the same cell line. In fact, structural variations of individual neoplastic cells on aging may lead to a heterogeneous data set, therefore falsifying confidence intervals, increasing error levels of analysis and consequently limiting the use of Raman spectroscopy analysis. We found that the observed morphological changes of U937 cells corresponded to well defined modifications of the Raman contributions in selected spectral regions, where markers of specific functional groups, useful to characterize the cell state, are present. A detailed subcellular analysis showed a change in cellular organization as a function of time, and correlated to a significant increase of apoptosis levels. Besides the aforementioned study, Raman spectra were used as input for principal component analysis (PCA) in order to detect and classify spectral changes among U937 cells.

A functional microRNA library screen reveals miR-410 as a novel anti-apoptotic regulator of cholangiocarcinoma.

PubMed

Palumbo, Tiziana; Poultsides, George A; Kouraklis, Grigorios; Liakakos, Theodore; Drakaki, Alexandra; Peros, George; Hatziapostolou, Maria; Iliopoulos, Dimitrios

2016-06-03

Cholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo. A functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors. The screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas. Taken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma.

Fabrication and characterization of solid oxide cells for energy conversion and storage

NASA Astrophysics Data System (ADS)

Yang, Chenghao

2011-12-01

There has been an increasing interest in clean and renewable energy generation for highlighted energy and environmental concerns. Solid oxide cells (SOCs) have been considered as one of the promising technologies, since they can be operated efficiently both in electrolysis mode by generating hydrogen through steam electrolysis and fuel cell mode by electrochemically combining fuel with oxidant. The present work is devoted to performing a fundamental study of SOC in both fuel cell mode for power generation and electrolysis mode for fuel production. The research work on SOCs that can be operated reversibly for power generation and fuel production has been conducted in the following six projects: (1) High performance solid oxide electrolysis cell (SOEC) Fabrication of novel structured SOEC oxygen electrode with the conventional and commercial solid oxide fuel cell materials by screen-printing and infiltration fabrication methods. The microstructure, electrochemical properties and durability of SOECs has been investigated. It was found that the LSM infiltrated cell has an area specific resistance (ASR) of 0.20 Ω cm2 at 900°C at open circuit voltage with 50% absolute humidity (AH), which is relatively lower than that of the cell with LSM-YSZ oxygen electrode made by a conventional mixing method. Electrolysis cell with LSM infiltrated oxygen electrode has demonstrated stable performance under electrolysis operation with 0.33 A/cm2 and 50 vol.% AH at 800°C. (2) Advanced performance high temperature micro-tubular solid oxide fuel cell (MT-SOFC) Phase-inversion, dip-coating, high temperature co-sintering process and impregnation method were used to fabricate micro-tubular solid oxide fuel cell. The micro-structure of the micro-tubular fuel cell will be investigated and the power output and thermal robustness has been evaluated. High performance and rapid start-up behavior have been achieved, indicates that the MT-SOFC developed in this work can be a promising technology for portable applications. (3) Promising intermediate temperature micro-tubular solid oxide fuel cells for portable power supply applications Maximum power densities of 0.5, 0.38 and 0.27 W/cm2 have been obtained using H2-15% H2O as fuel at 550, 600 and 650°C, respectively. Quick thermal cycles performed on the intermediate temperature MT-SOFC stability demonstrate that the cell has robust performance stability for portable applications. (4) Micro-tubular solid oxide cell (MT-SOC) for steam electrolysis The electrochemical properties of MT-SOC will be investigated in detail in electrolysis mode. The mechanism of the novel hydrogen electrode structure benefiting the cell performance will be demonstrated systematically. The high electrochemical performance of the MT-SOC in electrolysis mode indicates that MT-SOC can provide an efficient hydrogen generation process. (5) Micro-tubular solid oxide cell (MT-SOC) for steam and CO2 co-electrolysis The MT-SOC will be operated in co-electrolysis mode for steam and CO 2, which will provide an efficient approach to generate syngas (H2+CO) without consuming fossil fuels. This can potentially provide an alternative superior approach for carbon sequestration which has been a critical issue facing the sustainability of our society. (6) Steam and CO2 co-electrolysis using solid oxide cells fabricated by freeze-drying tape-casting Tri-layer scaffolds have been prepared by freeze-drying tape casting process and the electrode catalysts are obtained by infiltrating the porous electrode substrates. Button cells will be tested for co-electrolysis of steam and CO2. The mechanism and efficiency of steam and CO2 co-electrolysis will be systemically investigated. In conclusion, SOCs have been fabricated with conventional materials and evaluated, but their performance has been found to be limited in either SOFC or SOEC mode. The cell performance has been significantly improved by employing an infiltrated LSM-YSZ electrode, due to dramatically decreased polarization resistance. However, mass transport limitation has been observed, particularly in electrolysis mode. By utilizing micro-tubular SOCs with novel hydrogen electrode produced via a phase inversion method, mass transport limitation has been mitigated. Finally, mass transport has been further improved by using cells with electrodes fabricated through a freeze-drying tape-casting method. (Abstract shortened by UMI.)

Functional Dicer Is Necessary for Appropriate Specification of Radial Glia during Early Development of Mouse Telencephalon

PubMed Central

Nowakowski, Tomasz Jan; Mysiak, Karolina Sandra; Pratt, Thomas; Price, David Jonathan

2011-01-01

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon. PMID:21826226

Functional dicer is necessary for appropriate specification of radial glia during early development of mouse telencephalon.

PubMed

Nowakowski, Tomasz Jan; Mysiak, Karolina Sandra; Pratt, Thomas; Price, David Jonathan

2011-01-01

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1⁻/⁻ dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon.

Effects of silymarin and silymarin-doxorubicin applications on telomerase activity of human hepatocellular carcinoma cell line HepG2.

PubMed

Yurtcu, Erkan; Darcansoy Iseri, Ozlem; Iffet Sahin, Feride

2015-01-01

Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapeutics such as doxorubicin. Milk thistle extract, or its active constituent silymarin has been used by cancer patients as an alternative and complementary agent. Telomerase activation is one of the initial events of HCC. In this study, we applied doxorubicin and silymarin for 72 hrs in order to test individual and combined effect of the agents on telomerase activity. The effects of doxorubicin, silymarin, and their combination on the proliferation of HepG2 cell line were tested by MTT assay, and Checkerboard micro plate method was applied to define the nature of doxorubicin and silymarin interactions on the cells. Lipid peroxidations were assessed by thiobarbituric acid reactive substance (TBARS) level. Telomerase activity was determined according to the telomeric repeat amplification protocol (TRAP). Untreated cells were used as control group. Doxorubicin-silymarin combination had indifferent antiproliferative effects on HepG2 cells. Telomerase activity of the cells incubated with IC50 of doxorubicin and silymarin decreased to 72% (p<0.05). IC50 combinations of doxorubicin and silymarin caused 70% (p<0.05) reduction. All treatments except for the 1/2IC50 of silymarin caused significant increase in lipid peroxidation levels when compared to controls. TBARS levels did not significantly increase when doxorubicin and silymarin were applied in combination, which is in concordance with the indifferent drug interaction. IC50 of both doxorubicin and silymarin alone and in combination inhibited telomerase activity. Mechanism of inhibition may be elucidated by further molecular studies.

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

NASA Technical Reports Server (NTRS)

Allen, G. J.; Kwak, J. M.; Chu, S. P.; Llopis, J.; Tsien, R. Y.; Harper, J. F.; Schroeder, J. I.; Evans, M. L. (Principal Investigator)

1999-01-01

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.

Inhibition of MPP+-induced mitochondrial damage and cell death by trifluoperazine and W-7 in PC12 cells.

PubMed

Lee, Chung Soo; Park, Se Young; Ko, Hyun Hee; Song, Jin Ho; Shin, Yong Kyoo; Han, Eun Sook

2005-01-01

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of trifluoperazine and W-7 on the MPP+-induced mitochondrial damage and cell death in undifferentiated PC12 cells. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) at 0.5-1 microM significantly reduced the loss of cell viability in PC12 cells treated with 500 microM MPP+. Trifluoperazine and W-7 (0.5-1 microM) inhibited the nuclear damage, the loss of the mitochondrial transmembrane potential followed by cytochrome c release, and the elevation of intracellular Ca2+ levels due to MPP+ in PC12 cells and attenuated the formation of reactive oxygen species and the depletion of GSH. Calmodulin antagonists at 5-10 microM exhibited a cytotoxic effect on PC12 cells, and compounds at 10 microM did not attenuate cytotoxicity of MPP+. Calmodulin antagonists (0.5-1 microM) significantly reduced rotenone-induced mitochondrial damage and cell death, whereas they did not attenuate cell death and elevation of intracellular Ca2+ levels due to H2O2 or ionomycin. The results show that trifluoperazine and W-7 exhibit a differential inhibitory effect against cytotoxicity of MPP+ depending on concentration. Both compounds at the concentrations less than 5 microM may attenuate the MPP+-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering the intracellular Ca2+ levels.

Cell/surface interactions on laser micro-textured titanium-coated silicon surfaces.

PubMed

Mwenifumbo, Steven; Li, Mingwei; Chen, Jianbo; Beye, Aboubaker; Soboyejo, Wolé

2007-01-01

This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.

Simple Analysis of Lipid Inhibition Activity on an Adipocyte Micro-Cell Pattern Chip.

PubMed

Kim, Gi Yong; Yeom, Su-Jin; Jang, Sung-Chan; Lee, Chang-Soo; Roh, Changhyun; Jeong, Heon-Ho

2018-06-04

Polydimethyl-siloxane (PDMS) is often applied to fabricate cell chips. In this study, we fabricated an adipocyte microcell pattern chips using PDMS to analyze the inhibition activity of lipid droplets in mouse embryo fibroblast cells (3T3-L1) with anti-obesity agents. To form the PDMS based micropattern, we applied the micro-contact printing technique using PDMS micro-stamps that had been fabricated by conventional soft lithography. This PDMS micro-pattern enabled the selective growth of 3T3-L1 cells onto the specific region by preventing cell adhesion on the PDMS region. It then allowed growth of the 3T3-L1 cells in the chip for 10 days and confirmed that lipid droplets were formed in the 3T3-L1 cells. After treatment of orlistat and quercetin were treated in an adipocyte micro-cell pattern chip with 3T3-L1 cells for six days, we found that orlistat and quercetin exhibited fat inhibition capacities of 19.3% and 24.4% from 0.2 μM of lipid droplets in 3T3-L1 cells. In addition, we conducted a direct quantitative analysis of 3T3-L1 cell differentiation using Oil Red O staining. In conclusion, PDMS-based adipocyte micro-cell pattern chips may contribute to the development of novel bioactive compounds.

High-Throughput Mechanobiology Screening Platform Using Micro- and Nanotopography.

PubMed

Hu, Junqiang; Gondarenko, Alexander A; Dang, Alex P; Bashour, Keenan T; O'Connor, Roddy S; Lee, Sunwoo; Liapis, Anastasia; Ghassemi, Saba; Milone, Michael C; Sheetz, Michael P; Dustin, Michael L; Kam, Lance C; Hone, James C

2016-04-13

We herein demonstrate the first 96-well plate platform to screen effects of micro- and nanotopographies on cell growth and proliferation. Existing high-throughput platforms test a limited number of factors and are not fully compatible with multiple types of testing and assays. This platform is compatible with high-throughput liquid handling, high-resolution imaging, and all multiwell plate-based instrumentation. We use the platform to screen for topographies and drug-topography combinations that have short- and long-term effects on T cell activation and proliferation. We coated nanofabricated "trench-grid" surfaces with anti-CD3 and anti-CD28 antibodies to activate T cells and assayed for interleukin 2 (IL-2) cytokine production. IL-2 secretion was enhanced at 200 nm trench width and >2.3 μm grating pitch; however, the secretion was suppressed at 100 nm width and <0.5 μm pitch. The enhancement on 200 nm grid trench was further amplified with the addition of blebbistatin to reduce contractility. The 200 nm grid pattern was found to triple the number of T cells in long-term expansion, a result with direct clinical applicability in adoptive immunotherapy.

Pancreatic cancer: Translational research aspects and clinical implications

PubMed Central

Ansari, Daniel; Chen, Bi-Cheng; Dong, Lei; Zhou, Meng-Tao; Andersson, Roland

2012-01-01

Despite improvements in surgical techniques and adjuvant chemotherapy, the overall mortality rates in pancreatic cancer have generally remained relatively unchanged and the 5-year survival rate is actually below 2%. This paper will address the importance of achieving an early diagnosis and identifying markers for prognosis and response to therapy such as genes, proteins, microRNAs or epigenetic modifications. However, there are still major hurdles when translating investigational biomarkers into routine clinical practice. Furthermore, novel ways of secondary screening in high-risk individuals, such as artificial neural networks and modern imaging, will be discussed. Drug resistance is ubiquitous in pancreatic cancer. Several mechanisms of drug resistance have already been revealed, including human equilibrative nucleoside transporter-1 status, multidrug resistance proteins, aberrant signaling pathways, microRNAs, stromal influence, epithelial-mesenchymal transition-type cells and recently the presence of cancer stem cells/cancer-initiating cells. These factors must be considered when developing more customized types of intervention (“personalized medicine”). In the future, multifunctional nanoparticles that combine a specific targeting agent, an imaging probe, a cell-penetrating agent, a biocompatible polymer and an anti-cancer drug may become valuable for the management of patients with pancreatic cancer. PMID:22509073

In-situ monitoring of H2O2 degradation by live cells using voltammetric detection in a lab-on-valve system.

PubMed

Lähdesmäki, Ilkka; Park, Young K; Carroll, Andrea D; Decuir, Michael; Ruzicka, Jaromir

2007-08-01

This paper describes a method for monitoring the degradation of hydrogen peroxide by cells immobilized on a beaded support. The detection is based on the voltammetric reduction of hydrogen peroxide on a mercury film working electrode, whilst combining the concept of sequential injection (SI) with the lab-on-valve (LOV) manifold allows the measurements to be carried out in real time and automatically, in well-defined conditions. The method is shown to be capable of simultaneously monitoring hydrogen peroxide in the 10-1000 microM range and oxygen in the 160-616 microM range. A correction algorithm has been used to ensure reliable H2O2 results in the presence of varying oxygen levels. The method has been successfully applied to monitoring the degradation of H2O2 by wild-type cells and by catalase-overexpressing mouse embryonic fibroblasts. Since the technique allows the monitoring of the initial response rate, it provides data not accessible by current methods that are end-point-based measurements.

[Development of Sediment Micro-Interface Under Physical and Chironomus plumosus Combination Disturbance].

PubMed

Wang, Ren; Li, Da-peng; Huang, Yong; Liu, Yan-jian; Chen, Jun

2015-11-01

Synergistic effect of physical and Chironomus plumosus combination disturbance on the characteristics of the micro-environment and micro-interface was investigated by the Rhizon samplers and Unisense micro sensor system. The results showed that the oxygen penetration depth (OPD), total oxygen exchange (TOE), water content and total microbial activity increased under the combination disturbance and bioturbation and were kept at the higher level, compared with the control. These parameters increased with the physical intensity under combination disturbance. However, the content of Fe2+ decreased under the combination disturbance and bioturbation and the decrease was more obvious than that in the control. The changes of the Fe2+, the water content and the total microbial activity were large at 0-4 cm depth in the sediments. Therefore, the area might be the active area for the transformation of internal sedimentary phosphorus forms. The curve fitting was used for the OPD, TOE, the content of Fe2+, the water content and the total microbial activity with the physical intensity under combination disturbance. It was observed that the second-order polynomial equation was suitable for the curve fitting. In addition, jump type synergistic effect was presented in the above mentioned parameters under combination disturbance when the physical intensity was higher than 34 r x min(-1). The remodeling on the sediment micro-interface and micro-environment might be the main inducing mechanism for the transformation of internal phosphorus.

Identification of host miRNAs that may limit human rhinovirus replication

PubMed Central

Bondanese, Victor Paky; Francisco-Garcia, Ana; Bedke, Nicole; Davies, Donna E; Sanchez-Elsner, Tilman

2014-01-01

AIM: To test whether the replication of human rhinovirus (HRV) is regulated by microRNAs in human bronchial epithelial cells. METHODS: For the present study, the human cell line BEAS-2B (derived from normal human bronchial epithelial cells) was adopted. DICER knock-down, by siRNA transfection in BEAS-2B cells, was performed in order to inhibit microRNA maturation globally. Alternatively, antisense oligonucleotides (anti-miRs) were transfected to inhibit the activity of specific microRNAs. Cells were infected with HRV-1B. Viral replication was assessed by measuring the genomic viral RNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Association between microRNA-induced-silencing-complex and viral RNA was detected by Ago2 co-immunoprecipitation followed by RT-qPCR. Targetscan v.6 was used to predict microRNA target sites on several HRV strains. RESULTS: Here, we show that microRNAs affect replication of HRV-1B. DICER knock-down significantly reduced the expression of mature microRNAs in a bronchial epithelial cell line (BEAS-2B) and in turn, increased the synthesis of HRV-1B RNA. Additionally, HRV-1B RNA co-immunoprecipitated with argonaute 2 protein, an important effector for microRNA activity suggesting that microRNAs bind to viral RNA during infection. In order to identify specific microRNAs involved in this interaction, we employed bioinformatics analysis, and selected a group of microRNAs that have been reported to be under-expressed in asthmatic bronchial epithelial cells and were predicted to target different strains of rhinoviruses (HRV-1B, -16, -14, -27). Our results suggest that, out of this group of microRNAs, miR-128 and miR-155 contribute to the innate defense against HRV-1B: transfection of specific anti-miRs increased viral replication, as anticipated in-silico. CONCLUSION: Taken together, our results suggest that pathological changes in microRNA expression, as already reported for asthma or chronic obstructive pulmonary disease have the potential to affect Rhinovirus replication and therefore may play a role in virus-induced exacerbations. PMID:25426267

microRNA Profiling of Amniotic Fluid: Evidence of Synergy of microRNAs in Fetal Development.

PubMed

Sun, Tingting; Li, Weiyun; Li, Tianpeng; Ling, Shucai

2016-01-01

Amniotic fluid (AF) continuously exchanges molecules with the fetus, playing critical roles in fetal development especially via its complex components. Among these components, microRNAs are thought to be transferred between cells loaded in microvesicles. However, the functions of AF microRNAs remain unknown. To date, few studies have examined microRNAs in amniotic fluid. In this study, we employed miRCURY Locked Nucleotide Acid arrays to profile the dynamic expression of microRNAs in AF from mice on embryonic days E13, E15, and E17. At these times, 233 microRNAs were differentially expressed (p< 0.01), accounting for 23% of the total Mus musculus microRNAs. These differentially-expressed microRNAs were divided into two distinct groups based on their expression patterns. Gene ontology analysis showed that the intersectional target genes of these differentially-expressed microRNAs were mainly distributed in synapse, synaptosome, cell projection, and cytoskeleton. Pathway analysis revealed that the target genes of the two groups of microRNAs were synergistically enriched in axon guidance, focal adhesion, and MAPK signaling pathways. MicroRNA-mRNA network analysis and gene- mapping showed that these microRNAs synergistically regulated cell motility, cell proliferation and differentiation, and especially the axon guidance process. Cancer pathways associated with growth and proliferation were also enriched in AF. Taken together, the results of this study are the first to show the functions of microRNAs in AF during fetal development, providing novel insights into interpreting the roles of AF microRNAs in fetal development.

Fabrication of multi-scale periodic surface structures on Ti-6Al-4V by direct laser writing and direct laser interference patterning for modified wettability applications

NASA Astrophysics Data System (ADS)

Huerta-Murillo, D.; Aguilar-Morales, A. I.; Alamri, S.; Cardoso, J. T.; Jagdheesh, R.; Lasagni, A. F.; Ocaña, J. L.

2017-11-01

In this work, hierarchical surface patterns fabricated on Ti-6Al-4V alloy combining two laser micro-machining techniques are presented. The used technologies are based on nanosecond Direct Laser Writing and picosecond Direct Laser Interference Patterning. Squared shape micro-cells with different hatch distances were produced by Direct Laser Writing with depths values in the micro-scale, forming a well-defined closed packet. Subsequently, cross-like periodic patterns were fabricated by means of Direct Laser Interference Patterning using a two-beam configuration, generating a dual-scale periodic surface structure in both micro- and nano-scale due to the formation of Laser-Induced Periodic Surface Structure after the picosecond process. As a result a triple hierarchical periodic surface structure was generated. The surface morphology of the irradiated area was characterized with scanning electron microscopy and confocal microscopy. Additionally, static contact angle measurements were made to analyze the wettability behavior of the structures, showing a hydrophobic behavior for the hierarchical structures.

MicroRNA Expression Profile Selection for Cancer Staging Classification Using Backpropagation

NASA Astrophysics Data System (ADS)

Anjarwati; Wibowo, Adi; Adhy, Satriyo; Kusumaningrum, Retno

2018-05-01

Ovarian cancer, breast cancer, and lung cancer are deadly diseases and require serious treatment. The cancers are among the fifth most common causes of cancer-induced deaths especially for woman. The high mortality rate of cancer is caused by the lack of effective strategies for early detection of the cancer, whereas if its detected in the early stages, the life survival of cancer patients will be 90%, otherwise the survival rate only 30% when the cancers detected on metastasis stages or cancer cells have spread from a primary site of cancer. MicroRNAs can be used as potential biomarkers for cancer due to their profile expression on the cancers. In this paper, we proposed the feature selection of microRNA expression profiles for classification of the cancers stages using Backpropagation Neural Network. The Cancer stages are classified into before metastasis and after metastasis. Several combinations of the microRNA expression profiles from medical references are compared to find the best features for the classification. The accuracy and the mean square errors are used as basis testing the comparison.

Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro-dielectric barrier discharge plasma.

PubMed

Choi, Hyeongwon; Choi, Eun Ha; Kim, Kyung Sook

2017-10-01

Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro-dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro-DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro-DBD plasma for various exposure times. The elasticity of a single cell was determined by force-distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma-treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro-DBD plasma were related to cell mechanical changes. © 2017 Wiley Periodicals, Inc.

MicroRNA-26a Promotes Cholangiocarcinoma Growth by Activating β-catenin

PubMed Central

Zhang, Jinqiang; Han, Chang; Wu, Tong

2013-01-01

Background & Aims MicroRNAs (miRNAs) have been implicated in the development and progression of human cancers. We investigated the roles and mechanisms of miR-26a in human cholangiocarcinoma. Methods We used in situ hybridization and quantitative reverse transcriptase polymerase chain reaction to measure expression of miR-26a in human cholangiocarcinoma tissues and cell lines (eg, CCLP1, SG231, HuCCT1, TFK1). Human cholangiocarcinoma cell lines were transduced with lentiviruses that expressed miR-26a1 or a scrambled sequence (control); proliferation and colony formation were analyzed. We analyzed growth of human cholangiocarcinoma cells that overexpress miR-26a or its inhibitor in severe combined immune-deficient mice. Immunoblot, immunoprecipitation, DNA pull-down, immunofluorescence, and luciferase reporter assays were used to measure expression and activity of glycogen synthase kinase (GSK)-3β, β-catenin, and related signaling molecules. Results Human cholangiocarcinoma tissues and cell lines had increased levels of miR-26a compared with the noncancerous biliary epithelial cells. Overexpression of miR-26a increased proliferation of cholangiocarcinoma cells and colony formation in vitro, whereas miR-26 depletion reduced these parameters. In severe combined immune-deficient mice, overexpression of miR-26a by cholangiocarcinoma cells increased tumor growth and overexpression of the miR-26a inhibitor reduced it. GSK-3β messenger RNA was identified as a direct target of miR-26a by computational analysis and experimental assays. miR-26a–mediated reduction of GSK-3β resulted in activation of β-catenin and induction of several downstream genes including c-Myc, cyclinD1, and peroxisome proliferator-activated receptor δ. Depletion of β-catenin partially prevented miR-26a-induced tumor cell proliferation and colony formation. Conclusions miR-26a promotes cholangiocarcinoma growth by inhibition of GSK-3β and subsequent activation of β-catenin. These signaling molecules might be targets for prevention or treatment of cholangiocarcinoma. PMID:22484120

Epigenetic silencing of MicroRNA-503 regulates FANCA expression in non-small cell lung cancer cell.

PubMed

Li, Ning; Zhang, Fangfang; Li, Suyun; Zhou, Suzhen

2014-02-21

It is reported that MicroRNA-503 (miR-503) regulates cell apoptosis, and thus modulates the resistance of non-small cell lung cancer cells (NSCLC) to cisplatin. However, the exact role of miR-503 in NSCLC remains unknown. In the present study, the level of miR-503 expression in NSCLC was evaluated using realtime PCR, and the DNA methylation status within miR-503 promoter was analyzed by Combined Bisulfite Restriction Analysis (COBRA) or bisulfite-treated DNA sequencing assays (BSP). We found that the expression of miR-503 was significantly decreased in NSCLC tissues compared to normal tissues. A statistically significant inverse association was found between miR-503 methylation status and expression of the miR-503 in tumor tissues (P<0.001), and expression of miR-503 was restored by the demethylating agent 5-aza-2'-deoxycytidine, suggesting that methylation was associated with the transcriptional silencing. Then, we show that miR-503 targets a homologous DNA region in the 3'-UTR region of the Fanconi anemia complementation group A protein (FANCA) gene and represses its expression at the transcriptional level. Taken together, our results suggest that miR-503 regulates the resistance of non-small cell lung cancer cells to cisplatin at least in part by targeting FANCA. Copyright © 2014 Elsevier Inc. All rights reserved.

Engineering membrane scaffolds with both physical and biomolecular signaling.

PubMed

Tejeda-Montes, Esther; Smith, Katherine H; Poch, Marta; López-Bosque, María Jesús; Martín, Laura; Alonso, Matilde; Engel, Elisabeth; Mata, Alvaro

2012-03-01

We report on the combination of a top-down and bottom-up approach to develop thin bioactive membrane scaffolds based on functional elastin-like polymers (ELPs). Our strategy combines ELP cross-linking and assembly, and a variety of standard and novel micro/nanofabrication techniques to create self-supporting membranes down to ∼500 nm thick that incorporate both physical and biomolecular signals, which can be easily tailored for a specific application. In this study we used an ELP that included the cell-binding motif arginine-glycine-aspartic acid-serine (RGDS). Furthermore, fabrication processes were developed to create membranes that exhibited topographical patterns with features down to 200 nm in lateral dimensions and up to 10 μm in height on either one or both sides, uniform and well-defined pores, or multiple ELP layers. A variety of processing parameters were tested in order to optimize membrane fabrication, including ELP and cross-linker concentration, temperature, reaction time and ambient humidity. Membrane micro/nanopatterning, swelling and stiffness were characterized by atomic force microscopy, nanoindentation tests and scanning electron microscopy. Upon immersion in phosphate-buffered saline and an increase in temperature from 25 to 40°C, membranes exhibited a significant increase in surface stiffness, with the reduced Young's modulus increasing with temperature. Finally, rat mesenchymal stem cells were cultured on thin RGDS-containing membranes, which allowed cell adhesion, qualitatively enhanced spreading compared to membranes without RGDS epitopes and permitted proliferation. Furthermore, cell morphology was drastically affected by topographical patterns on the surface of the membranes. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

MicroRNA-873 mediates multidrug resistance in ovarian cancer cells by targeting ABCB1.

PubMed

Wu, Di-di; Li, Xue-Song; Meng, Xiao-Na; Yan, Jing; Zong, Zhi-Hong

2016-08-01

Ovarian cancer is commonly treated with cisplatin and paclitaxel combination chemotherapy; however, ovarian cancer cells often develop resistance to these drugs. Increasingly, microRNAs (miRNAs) including miR-873 have been implicated in drug resistance in many cancers, but the role of miR-873 in ovarian cancer remains unknown. MTT cell viability assays revealed that the sensitivities of ovarian cancer lines to cisplatin and paclitaxel increased following transfection with miR-873 (P < 0.05). After predicting the miR-873 binding region in the 3'-untranslated region of ABCB1, dual-luciferase reporter assay confirmed this prediction. RT-PCR and Western blotting revealed that MDR1 expression was significantly downregulated after transfection with miR-873 and upregulated after transfection with anti-miR-873 at both mRNA and protein levels compared to negative controls (P < 0.05). Experiments in a mouse xenograft model confirmed that intratumoral administration of miR-873 could enhance the efficacy of cisplatin in inhibiting tumor growth in ovarian cancer in vivo (P < 0.05). ABCB1 overexpression reduced sensitivities of ovarian cancer lines OVCAR3 and A2780 to cisplatin and paclitaxel, which can be reversed by miR-873 mimic transfection (P < 0.05). In summary, we demonstrated that overexpression of miR-873 increased the sensitivity of ovarian cancer cells to cisplatin and paclitaxel by targeting MDR1 expression. Our findings suggest that combination therapies with chemotherapy agents and miR-873 may suppress drug resistance in ovarian cancer.

Acrolein oxidizes the cytosolic and mitochondrial thioredoxins in human endothelial cells.

PubMed

Szadkowski, Adam; Myers, Charles R

2008-01-14

Acrolein is a reactive aldehyde that is a widespread environmental pollutant and can be generated endogenously from lipid peroxidation. The thioredoxin (Trx) system in endothelial cells plays a major role in the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, cells maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. In human microvascular endothelial cells, Trx1 was more sensitive than Trx2 to oxidation by acrolein. A 30-min exposure to 2.5 microM acrolein caused partial oxidation of Trx1 but not Trx2. The active site dithiol of Trx1 was essentially completely oxidized by 5 microM acrolein whereas 12.5 microM was required for complete oxidation of Trx2. Partial recovery of the Trx1 redox status was observed over a 4h acrolein-free recovery period, with increases in the reduced form and decreases in the fully oxidized form. For cells treated with 2.5 or 5 microM acrolein the recovery did not require protein synthesis, whereas protein synthesis was required for the return of reduced Trx1 in cells treated with 12.5 microM acrolein. Pretreatment of cells with N-acetylcysteine (NAC) resulted in partial protection of Trx1 from oxidation by acrolein. In cells treated with acrolein for 30 min, followed by a 14- to 16-h acrolein-free period, small but significant cytotoxic effects were observed with 2.5 microM acrolein whereas all cells were adversely affected by >or= 12.5 microM. NAC pretreatment significantly decreased the percentage of stressed cells subsequently exposed to 5 or 12.5 microM acrolein. Given the critical role of the thioredoxins in cell survival, the ability of acrolein to oxidize both thioredoxins should be taken into account for a thorough understanding of its cytotoxic effects.

A comparative study of the effect of Bio-Oss® in combination with concentrated growth factors or bone marrow-derived mesenchymal stem cells in canine sinus grafting.

PubMed

Wang, Fang; Li, Qiong; Wang, Zuolin

2017-08-01

To compare the effects of Bio-Oss ® in combination with concentrated growth factors (CGFs) and bone marrow-derived mesenchymal stem cells (BMSCs) on bone regeneration for maxillary sinus floor augmentation in beagle dogs. Six beagle dogs received bilateral maxillary sinus floor augmentation. Venous blood drawn from dogs was collected and centrifuged to obtain CGFs. BMSCs derived from canine bone marrow were cultured using density gradient centrifugation. The suspension of BMSCs was added onto Bio-Oss ® granules at a density of 2 × 10 6 cells/ml, and the BMSCs/Bio-Oss ® constructs were incubated for an additional 4 h before use. Twelve sinuses were grafted with a mixture of CGFs/Bio-Oss ® , BMSCs/Bio-Oss ® construct, or Bio-Oss ® alone. Six months later, the bone formation of bilateral sinuses was evaluated by Micro-CT, microhardness test, histological examination, and histomorphometry. No adverse effect was found in these dogs. The dome-shaped augmentation protruded into the sinus cavity. Micro-CT revealed that there was significant difference in BV/TV but not in Tb. N, between groups A, B, and C. The extent of microhardness in groups A and B was significantly higher than in group C. The proportion of newly formed bone in groups A and B showed significant difference when compared to group C (P ≤ 0.01). The amount of residual grafts in groups A and B was significantly lower than in group C. Grafting with Bio-Oss ® in combination with CGFs can increase new bone formation more efficiently than using Bio-Oss ® alone in a canine model. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.

PubMed

Winata, Patrick; Williams, Marissa; McGowan, Eileen; Nassif, Najah; van Zandwijk, Nico; Reid, Glen

2017-11-17

MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.

MiR-592 functions as a tumor suppressor in glioma by targeting IGFBP2.

PubMed

Peng, Tao; Zhou, Lixiang; Qi, Hui; Wang, Guangming; Luan, Yongxin; Zuo, Ling

2017-07-01

A growing body of evidence suggests that microRNA-592 is involved in tumor initiation and development in several types of human cancers. However, the biological functions and molecular mechanism of microRNA-592 in glioma remain unclear. In this study, we explored the potential role of microRNA-592 in glioma as well as the possible molecular mechanisms. Our results proved that microRNA-592 expression was significantly downregulated in glioma tissues and cell lines (p < 0.01). Functional assays revealed that overexpression of microRNA-592 dramatically reduced the cell proliferation, migration, and invasion and induced cell arrest at G1/G0 phase in vitro. Mechanistic investigations defined insulin-like growth factor binding protein 2 as a direct and functional downstream target of microRNA-592, which was involved in the microRNA-592-mediated tumor-suppressive effects in glioma cells. Moreover, the in vivo study showed that microRNA-592 overexpression produced the smaller tumor volume and weight in nude mice. In summary, these results elucidated the function of microRNA-592 in glioma progression and suggested a promising application of it in glioma treatment.

Three-dimensional electrospun polycaprolactone (PCL)/alginate hybrid composite scaffolds.

PubMed

Kim, Min Seong; Kim, GeunHyung

2014-12-19

Micro/nanofibrous scaffolds have been used widely in biomedical applications because the micro/nano-scale fibres resemble natural extracellular matrix and the high surface-to-volume ratio encourages cellular activities (attachment and proliferation). However, poor mechanical properties, low controllability of various shapes and difficulties in obtaining controllable pore structure have been obstacles to their use in hard-tissue regeneration. To overcome these shortcomings, we suggest a new composite system, which uses a combination method of wet electrospinning, rapid prototyping and a physical punching process. Using the process, we obtained polycaprolactone (PCL)/alginate composite scaffolds, consisting of electrospun PCL/alginate fibres and micro-sized PCL struts, with mean pore sizes of 821 ± 55 μm. To show the feasibility of the scaffolds for hard-tissue regeneration, the scaffolds were assessed not only for physical properties, including hydrophilicity, water absorption, and tensile and compressive strength, but also in vitro cellular responses (cell viability and proliferation) and osteogenic differentiation (alkaline phosphatase (ALP) activity, and mineralisation) by culturing with pre-osteoblasts (MC3T3-E1 cells). With the reinforcing micro-sized PCL struts, the elastic modulus of the PCL/alginate scaffold was significantly improved versus a pure PCL scaffold. Additionally, due to the alginate component in the fibrous scaffold, they showed significantly enhanced hydrophilic behaviour, water absorption (∼8-fold) and significant biological activities (∼1.6-fold for cell viability at 7 days, ∼2.3-fold for ALP activity at 14 days and ∼6.4-fold for calcium mineralisation at 14 days) compared with those of a pure PCL fibrous scaffold. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immunomodulation: A definitive role of microRNA-142.

PubMed

Sharma, Salil

2017-12-01

Majority of microRNAs are evolutionarily conserved in vertebrates. This is suggestive of their similar roles in regulation of gene networks. In addition to their conserved mature sequences and regulatory roles, a few microRNAs show very cell or tissue specific expression. These microRNAs are highly enriched in some cell types or organs. One such microRNA is microRNA-142 (miR-142). The classical stem-loop structure of miR142 encodes for two species of mature microRNAs; miR142-5p and miR142-3p. MiR-142 is abundant in cells of hematopoietic origin, and therefore, aptly plays a role in lineage differentiation of hematopoietic cells. Interestingly, over the years, miR-142 has gained considerable attention for its quintessential role in regulating immune response. This mini-review discusses the important functional roles of miR-142 in inflammatory and immune response in different physiological and disease setting. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishment of MicroRNA delivery system by PP7 bacteriophage-like particles carrying cell-penetrating peptide.

PubMed

Sun, Yanli; Sun, Yanhua; Zhao, Ronglan

2017-08-01

MicroRNAs have great therapeutic potential in cancer and other diseases. However, their instability and low in vivo delivery efficiency limits their application. Recombinant PP7 bacteriophage-based virus-like particles (VLPs) could protect microRNAs against rapid degradation by RNase by packaging specific exogenous pre-microRNAs using the pac site. Insertion of a cell-penetrating peptide (CPP) into the AB-loop of VLPs could significantly improve the delivery efficiency of microRNAs into mammalian cells. Unlike other microRNA delivery methods (viral or non-viral vectors), recombinant PP7 VLPs carrying a CPP and microRNA could be efficiently expressed in Escherichia coli using the one-plasmid double expression system. Here we showed that PP7 VLPs carrying a CPP penetrated hepatoma SK-HEP-1 cells and delivered the pre-microRNA-23b, which was processed into a mature product within 24 h; a concentration of 10 nM was sufficient for the inhibition of hepatoma cell migration via the downregulation of liver-intestine cadherin expression. Furthermore, PP7 VLPs carrying a CPP and a pre-microRNA were not infectious, replicative, or cytotoxic. Therefore, recombinant PP7 VLPs can be used for simultaneous and targeted delivery of both microRNAs and peptides because of their ability to package specific exogenous RNA using the pac site and to display peptides. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.

PubMed

Baumann, Pascal; Hahn, Tobias; Hubbuch, Jürgen

2015-10-01

Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance. © 2015 Wiley Periodicals, Inc.

A Combination RNAi-Chemotherapy Layer-by-Layer Nanoparticle for Systemic Targeting of KRAS/P53 with Cisplatin to Treat Non-small Cell Lung Cancer

PubMed Central

Gu, Li; Deng, Zhou J.; Roy, Sweta; Hammond, Paula T.

2017-01-01

Purpose Mutation of the Kirsten ras sarcoma viral oncogene homolog (KRAS) and loss of p53 function are commonly seen in non-small cell lung cancer (NSCLC). Combining therapeutics targeting these tumor defensive pathways with cisplatin in a single nanoparticle platform are rarely developed in clinic. Experimental Design Cisplatin was encapsulated in liposomes which multiple polyelectrolyte layers including siKRAS and miR-34a were built on to generate multifunctional layer-by-layer nanoparticle. Structure, size, and surface charge were characterized, in addition to in vitro toxicity studies. In vivo tumor targeting and therapy was investigated in an orthotopic lung cancer model by microCT, fluorescence imaging, and immunohistochemistry. Results The singular nanoscale formulation, incorporating oncogene siKRAS, tumor suppressor stimulating miR-34a, and cisplatin, has shown enhanced toxicity against lung cancer cell line, KP cell. In vivo, systemic delivery of the nanoparticles indicated a preferential uptake in lung of the tumor-bearing mice. Efficacy studies indicated prolonged survival of mice from the combination treatment. Conclusion The combination RNA-chemotherapy in an LbL formulation provides an enhanced treatment efficacy against NSCLC, indicating promising potential in clinic. PMID:28912139

Micro-PIXE study of Ag in digestive glands of a nano-Ag fed arthropod ( Porcellio scaber, Isopoda, Crustacea)

NASA Astrophysics Data System (ADS)

Tkalec, Živa Pipan; Drobne, Damjana; Vogel-Mikuš, Katarina; Pongrac, Paula; Regvar, Marjana; Štrus, Jasna; Pelicon, Primož; Vavpetič, Primož; Grlj, Nataša; Remškar, Maja

2011-10-01

Micro-proton induced X-ray emission (micro-PIXE) method was applied to study the micro-localization of silver (Ag) in digestive glands of a terrestrial arthropod (Porcellio scaber) after feeding on silver nanoparticles (nano-Ag) dosed food. The aim of our work was to assess whether feeding on nano-Ag results in the assimilation of silver (Ag) in digestive gland cells. To study micro-localization and elemental distribution of Ag, the animals were fed on food dosed with nanoparticles for 14 days under controlled laboratory conditions. At the end of the feeding exposure, the animals were dissected and digestive glands prepared for micro-PIXE analyses and TEM investigation. The results obtained by micro-PIXE documented high amounts of Ag inside S-cells of the digestive gland epithelium; however, TEM investigation did not show particle aggregates inside digestive gland cells. Also no adverse effect on feeding behavior was recorded what is a measure of toxic effects. We explain the presence of Ag inside the cells as a result of the assimilation of dissoluted Ag ions from ingested nano-Ag particles. Assimilation of excessive amounts of ingested metal ions in S-cells is a well known metal detoxification mechanism in isopods. We discuss the advantages of using micro-PIXE for the micro-localization of elements in biological tissue in studies of interactions between nanoparticles and biological systems.

Non-canonical microRNAs miR-320 and miR-702 promote proliferation in Dgcr8-deficient embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Byeong-Moo; Department of Medicine, Harvard Medical School, Boston, MA 02115; Choi, Michael Y., E-mail: mchoi@partners.org

2012-09-21

Highlights: Black-Right-Pointing-Pointer Embryonic stem cells (ESCs) lacking non-canonical miRNAs proliferate slower. Black-Right-Pointing-Pointer miR-320 and miR-702 are two non-canonical miRNAs expressed in ESCs. Black-Right-Pointing-Pointer miR-320 and miR-702 promote proliferation of Dgcr8-deficient ESCs. Black-Right-Pointing-Pointer miR-320 targets p57 and helps to release Dgcr8-deficient ESCs from G1 arrest. Black-Right-Pointing-Pointer miR-702 targets p21 and helps to release Dgcr8-deficient ESCs from G1 arrest. -- Abstract: MicroRNAs are known to contribute significantly to stem cell phenotype by post-transcriptionally regulating gene expression. Most of our knowledge of microRNAs comes from the study of canonical microRNAs that require two sequential cleavages by the Drosha/Dgcr8 heterodimer and Dicer to generatemore » mature products. In contrast, non-canonical microRNAs bypass the cleavage by the Drosha/Dgcr8 heterodimer within the nucleus but still require cytoplasmic cleavage by Dicer. The function of non-canonical microRNAs in embryonic stem cells (ESCs) remains obscure. It has been hypothesized that non-canonical microRNAs have important roles in ESCs based upon the phenotypes of ESC lines that lack these specific classes of microRNAs; Dicer-deficient ESCs lacking both canonical and non-canonical microRNAs have much more severe proliferation defect than Dgcr8-deficient ESCs lacking only canonical microRNAs. Using these cell lines, we identified two non-canonical microRNAs, miR-320 and miR-702, that promote proliferation of Dgcr8-deficient ESCs by releasing them from G1 arrest. This is accomplished by targeting the 3 Prime -untranslated regions of the cell cycle inhibitors p57 and p21 and thereby inhibiting their expression. This is the first report of the crucial role of non-canonical microRNAs in ESCs.« less

In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA

PubMed Central

La Rocca, Gaspare; Olejniczak, Scott H.; González, Alvaro J.; Briskin, Daniel; Vidigal, Joana A.; Spraggon, Lee; DeMatteo, Raymond G.; Radler, Megan R.; Lindsten, Tullia; Ventura, Andrea; Tuschl, Thomas; Leslie, Christina S.; Thompson, Craig B.

2015-01-01

MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3′ untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling. PMID:25568082

Characterization of the Tissue and Stromal Cell Components of Micro-Superficial Enhanced Fluid Fat Injection (Micro-SEFFI) for Facial Aging Treatment.

PubMed

Rossi, Martina; Roda, Barbara; Zia, Silvia; Vigliotta, Ilaria; Zannini, Chiara; Alviano, Francesco; Bonsi, Laura; Zattoni, Andrea; Reschiglian, Pierluigi; Gennai, Alessandro

2018-06-14

New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector ®, an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages. Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing.

Selective cytotoxic effect of non-thermal micro-DBD plasma

NASA Astrophysics Data System (ADS)

Kwon, Byung-Su; Choi, Eun Ha; Chang, Boksoon; Choi, Jeong-Hyun; Kim, Kyung Sook; Park, Hun-Kuk

2016-10-01

Non-thermal plasma has been extensively researched as a new cancer treatment technology. We investigated the selective cytotoxic effects of non-thermal micro-dielectric barrier discharge (micro-DBD) plasma in cervical cancer cells. Two human cervical cancer cell lines (HeLa and SiHa) and one human fibroblast (HFB) cell line were treated with micro-DBD plasma. All cells underwent apoptotic death induced by plasma in a dose-dependent manner. The plasma showed selective inhibition of cell proliferation in cervical cancer cells compared to HFBs. The selective effects of the plasma were also observed between the different cervical cancer cell lines. Plasma treatment significantly inhibited the proliferation of SiHa cells in comparison to HeLa cells. The changes in gene expression were significant in the cervical cancer cells in comparison to HFBs. Among the cancer cells, apoptosis-related genes were significantly enriched in SiHa cells. These changes were consistent with the differential cytotoxic effects observed in different cell lines.

A Theoretical Solid Oxide Fuel Cell Model for Systems Controls and Stability Design

NASA Technical Reports Server (NTRS)

Kopasakis, George; Brinson, Thomas; Credle, Sydni

2008-01-01

As the aviation industry moves toward higher efficiency electrical power generation, all electric aircraft, or zero emissions and more quiet aircraft, fuel cells are sought as the technology that can deliver on these high expectations. The hybrid solid oxide fuel cell system combines the fuel cell with a micro-turbine to obtain up to 70% cycle efficiency, and then distributes the electrical power to the loads via a power distribution system. The challenge is to understand the dynamics of this complex multidiscipline system and the design distributed controls that take the system through its operating conditions in a stable and safe manner while maintaining the system performance. This particular system is a power generation and a distribution system, and the fuel cell and micro-turbine model fidelity should be compatible with the dynamics of the power distribution system in order to allow proper stability and distributed controls design. The novelty in this paper is that, first, the case is made why a high fidelity fuel cell mode is needed for systems control and stability designs. Second, a novel modeling approach is proposed for the fuel cell that will allow the fuel cell and the power system to be integrated and designed for stability, distributed controls, and other interface specifications. This investigation shows that for the fuel cell, the voltage characteristic should be modeled but in addition, conservation equation dynamics, ion diffusion, charge transfer kinetics, and the electron flow inherent impedance should also be included.

Regulatory aspects of the glutamylation of methotrexate in cultured hepatoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nimec, Z.; Galivan, J.

1983-10-15

The glutamylation of methotrexate has been evaluated in H35 hepatoma cells in vitro as a function of the conditions of culture. Glutamylation yields methotrexate polyglutamate with two to five additional glutamate residues and is a saturable process. The rate of glutamylation increases little above 10 microM extracellular methotrexate which corresponds to an intracellular concentration of approximately 4 microM. The rate of glutamylation measured over a 6-h period was stimulated by a reduction in cellular folates and prior incubation of the cells with insulin. Glutamylation was also more rapid in dividing cultures than in confluent cells. The combination of insulin inclusionmore » and folate reduction, which was additive, caused approximately a fourfold increase in the rate of glutamylation over control cells under the conditions tested. The maximal rate of methotrexate glutamylation, which was 100 nmol/g/h, occurred in folate-depleted, insulin-supplemented cells. Supplementing folate-depleted cells with reduced folate coenzymes caused the glutamylation to be reduced by more than 90%. In addition to showing that folates can modify the rates of methotrexate polyglutamate formation, data are presented suggesting that methotrexate polyglutamates can regulate their own synthesis. The consequences of the formation of these retained forms of methotrexate in H35 hepatoma cells and the effects of potential regulators of this process are discussed in terms of the glutamylation of folates in the cells and the chemotherapeutic effects of antifolates.« less

microRNA Response to Listeria monocytogenes Infection in Epithelial Cells

PubMed Central

Izar, Benjamin; Mannala, Gopala Krishna; Mraheil, Mobarak Abu; Chakraborty, Trinad; Hain, Torsten

2012-01-01

microRNAs represent a family of very small non-coding RNAs that control several physiologic and pathologic processes, including host immune response and cancer by antagonizing a number of target mRNAs. There is limited knowledge about cell expression and the regulatory role of microRNAs following bacterial infections. We investigated whether infection with a Gram-positive bacterium leads to altered expression of microRNAs involved in the host cell response in epithelial cells. Caco-2 cells were infected with Listeria monocytogenes EGD-e, a mutant strain (ΔinlAB or Δhly) or incubated with purified listeriolysin (LLO). Total RNA was isolated and microRNA and target gene expression was compared to the expression in non-infected cells using microRNA microarrays and qRT-PCR. We identified and validated five microRNAs (miR- 146b, miR-16, let-7a1, miR-145 and miR-155) that were significantly deregulated following listerial infection. We show that expression patterns of particular microRNAs strongly depend on pathogen localization and the presence of bacterial effector proteins. Strikingly, miR-155 which was shown to have an important role in inflammatory responses during infection was induced by wild-type bacteria, by LLO-deficient bacteria and following incubation with purified LLO. It was downregulated following ΔinlAB infection indicating a new potent role for internalins in listerial pathogenicity and miRNA regulation. Concurrently, we observed differences in target transcript expression of the investigated miRNAs. We provide first evidence that L. monocytogenes infection leads to deregulation of a set of microRNAs with important roles in host response. Distinct microRNA expression depends on both LLO and pathogen localization. PMID:22312311

MSCs: The Sentinel and Safe-Guards of Injury.

PubMed

Caplan, Arnold I

2016-07-01

Mesenchymal stem cells (MSCs) were originally named because they could differentiate in a variety of mesenchymal phenotypes in culture. Evidence indicates that MSCs arise from perivascular cells, pericytes, when the blood vessels are broken or inflamed. These pericyte/MSCs are situated on every blood vessel in the body. The MSCs sense the micro-environment of the injury site and secrete site-specific factors that serve several important reparative functions: first, a curtain of molecules from the front of the MSCs provide a barrier from the interrogation of the over-aggressive immune system. Second, from the back of the MSCs, a different set of bioactive agents inhibit scar formation and establish a regenerative micro-environment. Third, if bacteria are sensed by the MSCs, they produce powerful protein antibiotics that kill the bacteria on contact. Last, the MSCs surround and encyst intruding solid objects like a piece of wood (a "splinter") or other foreign objects. The MSCs act as a combination paramedic and emergency room (ER) staff to survey the damage, isolate foreign components, stabilize the injured tissues, provide antibiotics and encysting protection before a slower, medicinal sequence can be initiated to regenerate the damaged tissue. The MSCs, thus, act as sentinels to safeguard the individual from intrusion and chronic injury. A societal treatment system has evolved, paramedics and ER procedures, which mirror in a macro-sense what MSCs orchestrate in a micro-sense. Key to this new understanding is that MSCs are not "stem cells," but rather as Medicinal Signaling Cells as the therapeutic agents. © 2015 Wiley Periodicals, Inc.

Methylation and microRNA-mediated epigenetic regulation of SOCS3

PubMed Central

Boosani, Chandra S.; Agrawal, Devendra K.

2017-01-01

Epigenetic gene silencing of several genes causes different pathological conditions in humans, and DNA methylation has been identified as one of the key mechanisms that underlie this evolutionarily conserved phenomenon associated with developmental and pathological gene regulation. Recent advances in the miRNA technology with high throughput analysis of gene regulation further increased our understanding on the role of miRNAs regulating multiple gene expression. There is increasing evidence supporting that the miRNAs not only regulate gene expression but they also are involved in the hypermethylation of promoter sequences, which cumulatively contributes to the epigenetic gene silencing. Here, we critically evaluated the recent progress on the transcriptional regulation of an important suppressor protein that inhibits cytokine-mediated signaling, SOCS3, whose expression is directly regulated both by promoter methylation and also by microRNAs, affecting its vital cell regulating functions. SOCS3 was identified as a potent inhibitor of Jak/STAT signaling pathway which is frequently upregulated in several pathologies, including cardiovascular disease, cancer, diabetes, viral infections, and the expression of SOCS3 was inhibited or greatly reduced due to hypermethylation of the CpG islands in its promoter region or suppression of its expression by different microRNAs. Additionally, we discuss key intracellular signaling pathways regulated by SOCS3 involving cellular events, including cell proliferation, cell growth, cell migration and apoptosis. Identification of the pathway intermediates as specific targets would not only aid in the development of novel therapeutic drugs, but, would also assist in developing new treatment strategies that could successfully be employed in combination therapy to target multiple signaling pathways. PMID:25682267

MicroRNA-7 Promotes Glycolysis to Protect against 1-Methyl-4-phenylpyridinium-induced Cell Death.

PubMed

Chaudhuri, Amrita Datta; Kabaria, Savan; Choi, Doo Chul; Mouradian, M Maral; Junn, Eunsung

2015-05-08

Parkinson disease is associated with decreased activity of the mitochondrial electron transport chain. This defect can be recapitulated in vitro by challenging dopaminergic cells with 1-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin that inhibits complex I of electron transport chain. Consequently, oxidative phosphorylation is blocked, and cells become dependent on glycolysis for ATP production. Therefore, increasing the rate of glycolysis might help cells to produce more ATP to meet their energy demands. In the present study, we show that microRNA-7, a non-coding RNA that protects dopaminergic neuronal cells against MPP(+)-induced cell death, promotes glycolysis in dopaminergic SH-SY5Y and differentiated human neural progenitor ReNcell VM cells, as evidenced by increased ATP production, glucose consumption, and lactic acid production. Through a series of experiments, we demonstrate that targeted repression of RelA by microRNA-7, as well as subsequent increase in the neuronal glucose transporter 3 (Glut3), underlies this glycolysis-promoting effect. Consistently, silencing Glut3 expression diminishes the protective effect of microRNA-7 against MPP(+). Further, microRNA-7 fails to prevent MPP(+)-induced cell death when SH-SY5Y cells are cultured in a low glucose medium, as well as when differentiated ReNcell VM cells or primary mouse neurons are treated with the hexokinase inhibitor, 2-deoxy-d-glucose, indicating that a functional glycolytic pathway is required for this protective effect. In conclusion, microRNA-7, by down-regulating RelA, augments Glut3 expression, promotes glycolysis, and subsequently prevents MPP(+)-induced cell death. This protective effect of microRNA-7 could be exploited to correct the defects in oxidative phosphorylation in Parkinson disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Automatic Detection and Evaluation of Solar Cell Micro-Cracks in Electroluminescence Images Using Matched Filters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spataru, Sergiu; Hacke, Peter; Sera, Dezso

A method for detecting micro-cracks in solar cells using two dimensional matched filters was developed, derived from the electroluminescence intensity profile of typical micro-cracks. We describe the image processing steps to obtain a binary map with the location of the micro-cracks. Finally, we show how to automatically estimate the total length of each micro-crack from these maps, and propose a method to identify severe types of micro-cracks, such as parallel, dendritic, and cracks with multiple orientations. With an optimized threshold parameter, the technique detects over 90 % of cracks larger than 3 cm in length. The method shows great potentialmore » for quantifying micro-crack damage after manufacturing or module transportation for the determination of a module quality criterion for cell cracking in photovoltaic modules.« less

The molecular mechanism of serum microRNA124b induced coronary heart disease by inducing myocardial cell senescence.

PubMed

Guo, M-L; Guo, L-L; Qin, Q-J; Weng, Y-Q; Wang, Y-N; Yao, J; Wang, Y-B; Zhang, X-Z; Ge, Z-M

2018-04-01

The incidence and mortality of coronary heart disease are rapidly increasing in recent years. Myocardial cell dysfunction and cell senescence may play a role in coronary heart disease. MicroRNA controls a variety of biological processes, but leaving its role in coronary heart disease has yet to be explored. Patients with coronary heart disease were regarded as subjects, and healthy volunteers as the control, on both of which microRNA124b level of serum was studied by Real-time PCR, and the heart function of patients was detected by using ultrasound. The relationship between serum microRNA124b level and cardiac function was analyzed along with the model of rat coronary artery disease; the level of aging proteins P21 and P53 in cardiac muscle cells was also tested. MicroRNA124b in the serum of patients with coronary heart disease was increased, and the heart function of patients was decreased (p < 0.05). Serum level of microRNA124b in a rat model of coronary heart disease was increased, and the cardiac function was decreased (p < 0.05). When myocardial cell appeared ageing, the level of P21 and P53 was increased, and the level of microRNA124b was related with P53. The level of microRNA124b in the serum of coronary heart disease patients and rat model may be related to the occurrence of coronary heart disease; microRNA124b may lead to the occurrence of coronary heart disease by causing cell senescence.

The effect of desferrioxamine on transferrin receptors, the cell cycle and growth rates of human leukaemic cells.

PubMed Central

Bomford, A; Isaac, J; Roberts, S; Edwards, A; Young, S; Williams, R

1986-01-01

The effect of the iron chelator, desferrioxamine, on transferrin binding, growth rates and the cell cycle was investigated in the human leukaemic cell line, K562. At all concentrations of the chelator (2-50 microM) binding of 125I-transferrin was increased by 24 h and reached a maximum at 72-96 h. Maximum binding (6-8-fold increased) occurred in cells treated with 20 microM-desferrioxamine, in contrast with control cells which, at 96 h, showed a 50% decrease over initial binding. Scatchard analysis at 4 degrees C showed that this increased binding was due to an increase in the number of receptors, as the Kd was similar in induced (1.8 nM) and control (1.5 nM) cells. After 96 h cells, cultured with 20 and 50 microM-desferrioxamine accumulated 59Fe from bovine transferrin at over twice the rate found with control cells, reflecting the increase in transferrin receptors. Although iron uptake was unimpaired by the chelator there was a dose-dependent inhibition of cell growth, with control cells completing three divisions in 96 h and those in 10 microM-desferrioxamine only two divisions. At the highest concentration (50 microM), cell division was abrogated although cell viability was maintained (85%). In contrast, DNA synthesis was not markedly affected, except at 50 microM-desferrioxamine when incorporation of [3H]thymidine was 52% of that in control cells. Flow cytometry revealed that there was a progressive accumulation of the cells in the active phases of their cycle (S, G2 + M). Desferrioxamine may increase transferrin receptors in two ways: by chelating a regulatory pool of iron within the cell, and by arresting cells in S phase when receptors are maximally expressed. PMID:3790074

Radiosensitization of tumour cell lines by the polyphenol Gossypol results from depressed double-strand break repair and not from enhanced apoptosis.

PubMed

Kasten-Pisula, Ulla; Windhorst, Sabine; Dahm-Daphi, Jochen; Mayr, Georg; Dikomey, Ekkehard

2007-06-01

New drugs are needed to increase the efficiency of radiotherapy in order to improve the therapeutic outcome of tumour patients. In this respect, the polyphenol Gossypol might be of interest, because of its effect on apoptosis and DNA repair, which is either mediated directly or indirectly via the inositol phosphate metabolism. It was investigated, whether these effects result in enhanced radiosensitivity of tumour cells. Tumour cell lines investigated: A549, FaDu, H1299, MCF7 and Du145. Cell cycle distribution was determined by FACS analysis, apoptosis was measured by DAPI staining and caspase3/7 activity. Double-strand breaks (DSB) were investigated via gammaH2AX-foci and cell survival by colony formation assay. The level of inositol phosphates was determined by HPLC, protein expression by Western blot. In A549 cells, Gossypol at concentrations 1microM strongly affects proliferation with only a modest arrest in the G1-phase, but with no increase in the fraction of apoptotic cells or the number of additional DSB. Additional DSB were only seen in FaDu cells, where Gossypol (2microM) was extremely toxic with a plating efficiency <0.002. When combined with irradiation, incubation with Gossypol (1-2microM) was found to result in an enhanced radiosensitivity with, however, a substantial variation. While there was a strong radiosensitization for FaDu and Du145 cells, there was an intermediate response for A549 cells, but almost no effect for H1299 and MCF7 cells. This sensitization was not caused from an elevated rate of apoptosis, but primarily resulted from reduced DSB repair capacity. The reduction in DSB repair could be ascribed neither to changes in the level of repair proteins relevant for non-homologous end-joining (Ku70, Ku80, DNA-PKcs) nor to changes in the level of higher phosphorylated inositols, whereby the latter were even found to be enhanced by Gossypol. For some tumour cell lines treatment with low concentrations of Gossypol can be used to inhibit DSB repair capacity and with that to increase the cellular radiosensitivity.

Effect of surface nano/micro-structuring on the early formation of microbial anodes with Geobacter sulfurreducens: Experimental and theoretical approaches.

PubMed

Champigneux, Pierre; Renault-Sentenac, Cyril; Bourrier, David; Rossi, Carole; Delia, Marie-Line; Bergel, Alain

2018-06-01

Smooth and nano-rough flat gold electrodes were manufactured with controlled Ra of 0.8 and 4.5nm, respectively. Further nano-rough surfaces (Ra 4.5nm) were patterned with arrays of micro-pillars 500μm high. All these electrodes were implemented in pure cultures of Geobacter sulfurreducens, under a constant potential of 0.1V/SCE and with a single addition of acetate 10mM to check the early formation of microbial anodes. The flat smooth electrodes produced an average current density of 0.9A·m -2 . The flat nano-rough electrodes reached 2.5A·m -2 on average, but with a large experimental deviation of ±2.0A·m -2 . This large deviation was due to the erratic colonization of the surface but, when settled on the surface, the cells displayed current density that was directly correlated to the biofilm coverage ratio. The micro-pillars considerably improved the experimental reproducibility by offering the cells a quieter environment, facilitating biofilm development. Current densities of up to 8.5A·m -2 (per projected surface area) were thus reached, in spite of rate limitation due to the mass transport of the buffering species, as demonstrated by numerical modelling. Nano-roughness combined with micro-structuring increased current density by a factor close to 10 with respect to the smooth flat surface. Copyright © 2018 Elsevier B.V. All rights reserved.

Universal lab-on-a-chip platform for complex, perfused 3D cell cultures

NASA Astrophysics Data System (ADS)

Sonntag, F.; Schmieder, F.; Ströbel, J.; Grünzner, S.; Busek, M.; Günther, K.; Steege, T.; Polk, C.; Klotzbach, U.

2016-03-01

The miniaturization, rapid prototyping and automation of lab-on-a-chip technology play nowadays a very important role. Lab-on-a-chip technology is successfully implemented not only for environmental analysis and medical diagnostics, but also as replacement of animals used for the testing of substances in the pharmaceutical and cosmetics industries. For that purpose the Fraunhofer IWS and partners developed a lab-on-a-chip platform for perfused cell-based assays in the last years, which includes different micropumps, valves, channels, reservoirs and customized cell culture modules. This technology is already implemented for the characterization of different human cell cultures and organoids, like skin, liver, endothelium, hair follicle and nephron. The advanced universal lab-on-a-chip platform for complex, perfused 3D cell cultures is divided into a multilayer basic chip with integrated micropump and application-specific 3D printed cell culture modules. Moreover a technology for surface modification of the printed cell culture modules by laser micro structuring and a complex and flexibly programmable controlling device based on an embedded Linux system was developed. A universal lab-on-a-chip platform with an optional oxygenator and a cell culture module for cubic scaffolds as well as first cell culture experiments within the cell culture device will be presented. The module is designed for direct interaction with robotic dispenser systems. This offers the opportunity to combine direct organ printing of cells and scaffolds with the microfluidic cell culture module. The characterization of the developed system was done by means of Micro-Particle Image Velocimetry (μPIV) and an optical oxygen measuring system.

Transcriptional regulation of development in heterocyst-forming cyanobacteria.

PubMed

Flores, Enrique; Picossi, Silvia; Valladares, Ana; Herrero, Antonia

2018-04-30

Filamentous, heterocyst-forming cyanobacteria are among the simplest multicellular systems in Nature. In the absence of combined nitrogen, the filaments consist of vegetative cells that fix CO 2 through oxygenic photosynthesis and micro-oxic heterocysts specialized for the fixation of N 2 in a proportion of about 10 to 1. The development of a heterocyst-containing filament involves differentiation of vegetative cells into heterocysts in a process that requires a distinct gene expression program. Two transcription factors are strictly required, NtcA and HetR. The CRP-family protein NtcA directly activates the expression of multiple genes during heterocyst differentiation - in some cases assisted by coactivators including HetR - and in mature heterocysts, whereas HetR is needed to build high NtcA levels in differentiating heterocysts and directly activates some particular genes. A few other regulators of gene expression participate at specific differentiation steps, and a specific transcription factor, CnfR, activates nif gene expression under the micro-oxic conditions of the heterocyst. Copyright © 2018 Elsevier B.V. All rights reserved.

Mechanisms of hypochlorite injury of target cells.

PubMed Central

Schraufstätter, I U; Browne, K; Harris, A; Hyslop, P A; Jackson, J H; Quehenberger, O; Cochrane, C G

1990-01-01

HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants. PMID:2153710

On the micro-indentation of plant cells in a tissue context

NASA Astrophysics Data System (ADS)

Mosca, Gabriella; Sapala, Aleksandra; Strauss, Soeren; Routier-Kierzkowska, Anne-Lise; Smith, Richard S.

2017-02-01

The effect of geometry on cell stiffness measured with micro-indentation techniques has been explored in single cells, however it is unclear if results on single cells can be readily transferred to indentation experiments performed on a tissue in vivo. Here we explored this question by using simulation models of osmotic treatments and micro-indentation experiments on 3D multicellular tissues with the finite element method. We found that the cellular context does affect measured cell stiffness, and that several cells of context in each direction are required for optimal results. We applied the model to micro-indentation data obtained with cellular force microscopy on the sepal of A. thaliana, and found that differences in measured stiffness could be explained by cellular geometry, and do not necessarily indicate differences in cell wall material properties or turgor pressure.

MicroRNA and extracellular vesicles in glioblastoma – Small but powerful

PubMed Central

Rooj, Arun K.; Mineo, Marco; Godlewski, Jakub

2016-01-01

To promote the tumor growth, angiogenesis, metabolism, and invasion, glioblastoma multiforme (GBM) cells subvert the surrounding microenvironment by influencing the endogenous activity of other brain cells including endothelial cells, macrophages, astrocytes, and microglia. Large number of studies indicates that the intracellular communication between the different cell types of the GBM microenvironment occurs through the functional transfer of oncogenic components such as proteins, non-coding RNAs, DNA and lipids via the release and uptake of extracellular vesicles (EVs). Unlike the communication through the secretion of chemokines and cytokines, the transfer and gene silencing activity of microRNAs through EVs is more complex as the biogenesis and proper packaging of microRNAs is crucial for their uptake by recipient cells. Although the specific mechanism of EV-derived microRNA uptake and processing in recipient cells is largely unknown, the screening, identifying and finally targeting of the EV-associated pro-tumorigenic microRNAs are emerging as new therapeutic strategy to combat the GBM. PMID:26968172

CHO microRNA engineering is growing up: Recent successes and future challenges☆

PubMed Central

Jadhav, Vaibhav; Hackl, Matthias; Druz, Aliaksandr; Shridhar, Smriti; Chung, Cheng-Yu; Heffner, Kelley M.; Kreil, David P.; Betenbaugh, Mike; Shiloach, Joseph; Barron, Niall; Grillari, Johannes; Borth, Nicole

2013-01-01

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines. PMID:23916872

In-vitro micro-Raman study of tissue samples for detecting cervical and ovarian cancer with 785-nm laser excitation

NASA Astrophysics Data System (ADS)

Sharma, S. K.; Kamemoto, L. E.; Misra, A. K.; Goodman, M. T.; Luk, H. W.; Killeen, J. L.

2010-04-01

We present results of in vitro micro-Raman spectroscopy of normal and cancerous cervical and ovarian tissues excited with 785 nm near-infrared (NIR) laser. Micro- Raman spectra of squamous cervical cells of both cervix and ovarian tissues show significant differences in the spectra of normal and cancerous cells. In particular, several well-defined Raman peaks in the 775-975 cm-1 region are observed in the spectra of normal cervix squamous cells but are completely missing in the spectra of invasive cervical cancer cells. In the high-frequency 2800-3100 cm-1 region it is shown that the peak area under CH stretching band is much lower than the corresponding area in the spectra of normal cells. In the case of ovarian tissues, the micro-Raman spectra show noticeable spectral differences between normal cells and ovarian serous cancer cells. In particular, we observed the accumulation of β-carotene in ovarian serous cancer cells compared to normal ovarian cells from women with no ovarian cancer. The NIR micro-Raman spectroscopy offers a potential molecular technique for detecting cervical and ovarian cancer from the respective tissues.

Reversible Block of Mouse Neural Stem Cell Differentiation in the Absence of Dicer and MicroRNAs

PubMed Central

Sansom, Stephen N.; Alsiö, Jessica M.; Kaneda, Masahiro; Smith, James; O'Carroll, Donal; Tarakhovsky, Alexander; Livesey, Frederick J.

2010-01-01

Background To investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs. Principal Findings Dicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia. Conclusions The block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation. PMID:20976144

Acetylcholine, carbachol, and GABA induce no detectable change in the length of isolated outer hair cells.

PubMed

Bobbin, R P; Fallon, M; Puel, J L; Bryant, G; Bledsoe, S C; Zajic, G; Schacht, J

1990-08-01

The mechanical and electrical properties of cochlear outer hair cells (OHCs) are suggested to modulate transduction by inner hair cells. These properties of OHCs are presumably regulated by efferent neurons which use several transmitters including acetylcholine (Ach) and gamma aminobutyric acid (GABA). Since it had been suggested that Ach causes isolated OHCs to shorten visibly, this study was designed to investigate whether GABA also alters the length of OHCs. OHCs were isolated from the guinea pig cochlea by mechanical dispersion after collagenase treatment. Cells were initially selected by strict morphological criteria. In addition they were only included in further studies if they attained a constant length during 10 min of superfusion with buffer solution. Neither GABA (20 microM: 100 microM), Ach (5 mM; 10 microM with 10 microM eserine) or carbachol (10 microM; 100 microM) altered OHC length when applied in iso-osmotic Hank's balanced salt solution (total number of cells tested, 72). If a change in length occurred it must have been smaller than 0.3 microns, our detection ability. In contrast, high potassium and variations in osmolarity changed hair cell length by 3-10% in agreement with other reports.

A microfluidic fuel cell with flow-through porous electrodes.

PubMed

Kjeang, Erik; Michel, Raphaelle; Harrington, David A; Djilali, Ned; Sinton, David

2008-03-26

A microfluidic fuel cell architecture incorporating flow-through porous electrodes is demonstrated. The design is based on cross-flow of aqueous vanadium redox species through the electrodes into an orthogonally arranged co-laminar exit channel, where the waste solutions provide ionic charge transfer in a membraneless configuration. This flow-through architecture enables improved utilization of the three-dimensional active area inside the porous electrodes and provides enhanced rates of convective/diffusive transport without increasing the parasitic loss required to drive the flow. Prototype fuel cells are fabricated by rapid prototyping with total material cost estimated at 2 USD/unit. Improved performance as compared to previous microfluidic fuel cells is demonstrated, including power densities at room temperature up to 131 mW cm-2. In addition, high overall energy conversion efficiency is obtained through a combination of relatively high levels of fuel utilization and cell voltage. When operated at 1 microL min-1 flow rate, the fuel cell produced 20 mW cm-2 at 0.8 V combined with an active fuel utilization of 94%. Finally, we demonstrate in situ fuel and oxidant regeneration by running the flow-through architecture fuel cell in reverse.

Morphological Characterisation of Unstained and Intact Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography

NASA Astrophysics Data System (ADS)

Walton, Lucy A.; Bradley, Robert S.; Withers, Philip J.; Newton, Victoria L.; Watson, Rachel E. B.; Austin, Clare; Sherratt, Michael J.

2015-05-01

Characterisation and quantification of tissue structures is limited by sectioning-induced artefacts and by the difficulties of visualising and segmenting 3D volumes. Here we demonstrate that, even in the absence of X-ray contrast agents, X-ray computed microtomography (microCT) and nanotomography (nanoCT) can circumvent these problems by rapidly resolving compositionally discrete 3D tissue regions (such as the collagen-rich adventitia and elastin-rich lamellae in intact rat arteries) which in turn can be segmented due to their different X-ray opacities and morphologies. We then establish, using X-ray tomograms of both unpressurised and pressurised arteries that intra-luminal pressure not only increases lumen cross-sectional area and straightens medial elastic lamellae but also induces profound remodelling of the adventitial layer. Finally we apply microCT to another human organ (skin) to visualise the cell-rich epidermis and extracellular matrix-rich dermis and to show that conventional histological and immunohistochemical staining protocols are compatible with prior X-ray exposure. As a consequence we suggest that microCT could be combined with optical microscopy to characterise the 3D structure and composition of archival paraffin embedded biological materials and of mechanically stressed dynamic tissues such as the heart, lungs and tendons.

Morphological Characterisation of Unstained and Intact Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography

PubMed Central

Walton, Lucy A.; Bradley, Robert S.; Withers, Philip J.; Newton, Victoria L.; Watson, Rachel E. B.; Austin, Clare; Sherratt, Michael J.

2015-01-01

Characterisation and quantification of tissue structures is limited by sectioning-induced artefacts and by the difficulties of visualising and segmenting 3D volumes. Here we demonstrate that, even in the absence of X-ray contrast agents, X-ray computed microtomography (microCT) and nanotomography (nanoCT) can circumvent these problems by rapidly resolving compositionally discrete 3D tissue regions (such as the collagen-rich adventitia and elastin-rich lamellae in intact rat arteries) which in turn can be segmented due to their different X-ray opacities and morphologies. We then establish, using X-ray tomograms of both unpressurised and pressurised arteries that intra-luminal pressure not only increases lumen cross-sectional area and straightens medial elastic lamellae but also induces profound remodelling of the adventitial layer. Finally we apply microCT to another human organ (skin) to visualise the cell-rich epidermis and extracellular matrix-rich dermis and to show that conventional histological and immunohistochemical staining protocols are compatible with prior X-ray exposure. As a consequence we suggest that microCT could be combined with optical microscopy to characterise the 3D structure and composition of archival paraffin embedded biological materials and of mechanically stressed dynamic tissues such as the heart, lungs and tendons. PMID:25975937

Scriptaid overcomes hypoxia-induced cisplatin resistance in both wild-type and mutant p53 lung cancer cells

PubMed Central

Pradhan, Shrikant; Mahajan, Divyank; Kaur, Prabhjot; Pandey, Namita; Sharma, Chandresh; Srivastava, Tapasya

2016-01-01

Non-small cell lung cancer (NSCLC), comprising 85% of lung cancer cases, has been associated with resistance to chemo/radiotherapy. The hypoxic tumor micro-environment, where insufficient vasculature results in poor drug penetrance and sub-optimal chemotherapy in the tumor interiors contributes heavily to this resistance. Additionally, epigenetic changes in tumorigenic cells also change their response to different forms of therapy. In our study, we have investigated the effectiveness of a combination of cisplatin with scriptaid [a pan-Histone Deacetylase inhibitor (HDACi)] in a model that mimics the tumor microenvironment of hypoxia and sub-lethal chemotherapy. Scriptaid synergistically increases the efficacy of cisplatin in normoxia as well as hypoxia, accompanied with reduced metastasis and enhanced DNA damage. Addition of scriptaid also overcomes the cisplatin resistance exhibited in lung cancer cells with stabilized hypoxia inducible factor 1 (HIF1)-α (mutant) and mutant p53. Molecular studies showed that the combination treatment increased apoptotic cell death in both normoxia and hypoxia with a dual role of p38MAPK. Together, our results suggest that the combination of low dose cisplatin and scriptaid is cytotoxic to NSCLC lines, can overcome hypoxia induced resistance and mutant p53- induced instability often associated with this cancer, and has the potential to be an effective therapeutic modality. PMID:27708247

Combined Multidimensional Microscopy as a Histopathology Imaging Tool.

PubMed

Shami, Gerald J; Cheng, Delfine; Braet, Filip

2017-02-01

Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy, thus, facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterize various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high-resolution backscattered scanning electron microscopy, we were able to further characterize the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. J. Cell. Physiol. 232: 249-256, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The MicroActive project: automatic detection of disease-related molecular cell activity

NASA Astrophysics Data System (ADS)

Furuberg, Liv; Mielnik, Michal; Johansen, Ib-Rune; Voitel, Jörg; Gulliksen, Anja; Solli, Lars; Karlsen, Frank; Bayer, Tobias; Schönfeld, Friedhelm; Drese, Klaus; Keegan, Helen; Martin, Cara; O'Leary, John; Riegger, Lutz; Koltay, Peter

2007-05-01

The aim of the MicroActive project is to develop an instrument for molecular diagnostics. The instrument will first be tested for patient screening for a group of viruses causing cervical cancer. Two disposable polymer chips with reagents stored on-chip will be inserted into the instrument for each patient sample. The first chip performs sample preparation of the epithelial cervical cells while mRNA amplification and fluorescent detection takes place in the second chip. More than 10 different virus markers will be analysed in one chip. We report results on sub-functions of the amplification chip. The sample is split into smaller droplets, and the droplets move in parallel channels containing different dried reagents for the different analyses. We report experimental results on parallel droplet movement control using one external pump only, combined with hydrophobic valves. Valve burst pressures are controlled by geometry. We show droplet control using valves with burst pressures between 800 and 4500 Pa. We also monitored the re-hydration times for two necessary dried reagents. After sample insertion, uniform concentration of the reagents in the droplet was reached after respectively 60 s and 10 min. These times are acceptable for successful amplification. Finally we have shown positive amplification of HPV type 16 using dried enzymes stored in micro chambers.

Enhanced osseointegration of titanium implants in a rat model of osteoporosis using multilayer bone mesenchymal stem cell sheets

PubMed Central

Duan, Yan; Ma, Wei; Li, Dehua; Wang, Tongfei; Liu, Baolin

2017-01-01

The present study aimed to investigate whether bone marrow-derived mesenchymal stem cell (BMSC) sheets combined with titanium implants enhanced implant osseointegration in an ovariectomized (OVX) rat model of osteoporosis. Sprague-Dawley rats were randomly assigned into a test group and control group. Allogenic BMSCs were collected from the rats, cultured and stored via cryopreservation. At 6 months post-ovariectomy, establishment of the OVX model was confirmed by micro-computed tomography (CT) measurements. BMSC sheets were subsequently layered and wrapped over titanium implants for implantation. Unmodified implants served as the control. At 8 weeks post-implantation, samples were observed by micro-CT reconstruction and histomorphometric evaluation. Micro-CT reconstruction identified a marked improvement in the surrounding bone volume following treatment, with data analyses indicating a significant increase in bone volume in the BMSC-implant group compared with the control implant group (P<0.05). In addition, histological staining identified new bone formation and an increased rate of bone-implant contact surrounding the BMSC-implant constructs. These results indicate that the use of BMSC sheets as a novel tissue engineering approach improves the osseointegration of titanium implants in an osteoporosis model. This method may expand the operative indications in patients with osteoporosis and improve the success rate of clinical dental implant treatments. PMID:29250137

Adhesion and proliferation of OCT-1 osteoblast-like cells on micro- and nano-scale topography structured poly(L-lactide).

PubMed

Wan, Yuqing; Wang, Yong; Liu, Zhimin; Qu, Xue; Han, Buxing; Bei, Jianzhong; Wang, Shenguo

2005-07-01

The impact of the surface topography of polylactone-type polymer on cell adhesion was to be concerned because the micro-scale texture of a surface can provide a significant effect on the adhesion behavior of cells on the surface. Especially for the application of tissue engineering scaffold, the pore size could have an influence on cell in-growth and subsequent proliferation. Micro-fabrication technology was used to generate specific topography to investigate the relationship between the cells and surface. In this study the pits-patterned surfaces of polystyrene (PS) film with diameters 2.2 and 0.45 microm were prepared by phase-separation, and the corresponding scale islands-patterned PLLA surface was prepared by a molding technique using the pits-patterned PS as a template. The adhesion and proliferation behavior of OCT-1 osteoblast-like cells morphology on the pits- and islands-patterned surface were characterized by SEM observation, cell attachment efficiency measurement and MTT assay. The results showed that the cell adhesion could be enhanced on PLLA and PS surface with nano-scale and micro-scale roughness compared to the smooth surfaces of the PLLA and PS. The OCT-1 osteoblast-like cells could grow along the surface with two different size islands of PLLA and grow inside the micro-scale pits of the PS. However, the proliferation of cells on the micro- and nano-scale patterned surface has not been enhanced compared with the controlled smooth surface.

Protective effects of anethole dithiolethione against oxidative stress-induced cytotoxicity in human Jurkat T cells.

PubMed

Khanna, S; Sen, C K; Roy, S; Christen, M O; Packer, L

1998-07-01

The protective effects of anethole dithiolethione (ADT) against H2O2- or 4-hydroxynonenal (HNE)-induced cytotoxicity in human Jurkat T cells were investigated. Jurkat T cells were pretreated with ADT (10-50 microM) for 18 hr and then challenged with H202 or HNE for up to 4 hr. Cytotoxicity was assessed by measuring: 1) leakage of lactate dehydrogenase from cells to medium; and 2) exclusion of the DNA intercalating fluorescent probe propidium iodide by viable cells. Pretreatment of cells with ADT (10 or 25 microM) for 18 hr significantly protected cells against H202- or HNE-induced cytotoxicity. Treatment of cells with ADT (10-50 microM) for 72 hr significantly increased the activities of catalase and glutathione reductase. The maximum effect of ADT treatment on the activity of these enzymes was observed when cells were treated with 25 microM of ADT for 72 hr. A significant increase in cellular GSH was observed in cells that were treated with ADT for 72 hr. Using monobromobimane as a thiol probe, we consistently observed that cells pretreated for 18 hr with ADT (25 or 50 microM) had also increased total thiol content. Exposure of Jurkat T cells to H202 or HNE resulted in a time-dependent decrease in cellular GSH. ADT (10-50 microM, 18 hr) pretreatment circumvented H202-dependent lowering of cellular GSH. In conclusion, ADT proved to be a potent cytoprotective thiol antioxidant with multifaceted mechanisms of action, suggesting that the drug has a remarkable therapeutic potential.

Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

PubMed

Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

2017-12-21

High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

MICRODISSECTION TESTICULAR SPERM EXTRACTION IN MEN WITH SERTOLI CELL ONLY TESTICULAR HISTOLOGY

PubMed Central

Berookhim, Boback M.; Palermo, Gianpiero D.; Zaninovic, Nikica; Rosenwaks, Zev; Schlegel, Peter N.

2015-01-01

Objective To study the outcomes of microdissection testicular sperm extraction (microTESE) among men with pure Sertoli cell only histology on diagnostic testicular biopsy. Design Retrospective cohort study. Setting Tertiary referral center. Patients 640 patients with pure Sertoli cell only histology on testicular biopsy who underwent microTESE by a single surgeon. Intervention MicroTESE. Main Outcome Measure Sperm retrieval rates. Results Overall, 44.5% of patients with Sertoli cell-only had sperm retrieved with microTESE. No difference was noted in sperm retrieval rates based on testis volume (≥ 15cc versus <15cc, 35.3% versus 46.1%, respectively). Patients with ≥ 15cc testicular volume and FSH 10-15 mU/mL had the worst prognosis, with a sperm retrieval rate of 6.7%. Conclusions Patients with previous testicular biopsy demonstrating Sertoli cell only histology can be counseled that they have a reasonable likelihood of sperm retrieval with the contemporary delivery of microTESE. Given this finding, the utility of testicular biopsy prior to microTESE is further questioned. PMID:25441063

Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

PubMed Central

Murphy, Patricia; Kabir, Md Humayun; Srivastava, Tarini; Mason, Michele E.; Dewi, Chitra U.; Lim, Seakcheng; Yang, Andrian; Djordjevic, Djordje; Killingsworth, Murray C.; Ho, Joshua W. K.; Harman, David G.

2018-01-01

ABSTRACT Cataracts cause vision loss and blindness by impairing the ability of the ocular lens to focus light onto the retina. Various cataract risk factors have been identified, including drug treatments, age, smoking and diabetes. However, the molecular events responsible for these different forms of cataract are ill-defined, and the advent of modern cataract surgery in the 1960s virtually eliminated access to human lenses for research. Here, we demonstrate large-scale production of light-focusing human micro-lenses from spheroidal masses of human lens epithelial cells purified from differentiating pluripotent stem cells. The purified lens cells and micro-lenses display similar morphology, cellular arrangement, mRNA expression and protein expression to human lens cells and lenses. Exposing the micro-lenses to the emergent cystic fibrosis drug Vx-770 reduces micro-lens transparency and focusing ability. These human micro-lenses provide a powerful and large-scale platform for defining molecular disease mechanisms caused by cataract risk factors, for anti-cataract drug screening and for clinically relevant toxicity assays. PMID:29217756

Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro.

PubMed

Murphy, Patricia; Kabir, Md Humayun; Srivastava, Tarini; Mason, Michele E; Dewi, Chitra U; Lim, Seakcheng; Yang, Andrian; Djordjevic, Djordje; Killingsworth, Murray C; Ho, Joshua W K; Harman, David G; O'Connor, Michael D

2018-01-09

Cataracts cause vision loss and blindness by impairing the ability of the ocular lens to focus light onto the retina. Various cataract risk factors have been identified, including drug treatments, age, smoking and diabetes. However, the molecular events responsible for these different forms of cataract are ill-defined, and the advent of modern cataract surgery in the 1960s virtually eliminated access to human lenses for research. Here, we demonstrate large-scale production of light-focusing human micro-lenses from spheroidal masses of human lens epithelial cells purified from differentiating pluripotent stem cells. The purified lens cells and micro-lenses display similar morphology, cellular arrangement, mRNA expression and protein expression to human lens cells and lenses. Exposing the micro-lenses to the emergent cystic fibrosis drug Vx-770 reduces micro-lens transparency and focusing ability. These human micro-lenses provide a powerful and large-scale platform for defining molecular disease mechanisms caused by cataract risk factors, for anti-cataract drug screening and for clinically relevant toxicity assays. © 2018. Published by The Company of Biologists Ltd.

Micro-Tubular Fuel Cells

NASA Technical Reports Server (NTRS)

Kimble, Michael C.; Anderson, Everett B.; Jayne, Karen D.; Woodman, Alan S.

2004-01-01

Micro-tubular fuel cells that would operate at power levels on the order of hundreds of watts or less are under development as alternatives to batteries in numerous products - portable power tools, cellular telephones, laptop computers, portable television receivers, and small robotic vehicles, to name a few examples. Micro-tubular fuel cells exploit advances in the art of proton-exchange-membrane fuel cells. The main advantage of the micro-tubular fuel cells over the plate-and-frame fuel cells would be higher power densities: Whereas the mass and volume power densities of low-pressure hydrogen-and-oxygen-fuel plate-and-frame fuel cells designed to operate in the targeted power range are typically less than 0.1 W/g and 0.1 kW/L, micro-tubular fuel cells are expected to reach power densities much greater than 1 W/g and 1 kW/L. Because of their higher power densities, micro-tubular fuel cells would be better for powering portable equipment, and would be better suited to applications in which there are requirements for modularity to simplify maintenance or to facilitate scaling to higher power levels. The development of PEMFCs has conventionally focused on producing large stacks of cells that operate at typical power levels >5 kW. The usual approach taken to developing lower-power PEMFCs for applications like those listed above has been to simply shrink the basic plate-and-frame configuration to smaller dimensions. A conventional plate-and-frame fuel cell contains a membrane/electrode assembly in the form of a flat membrane with electrodes of the same active area bonded to both faces. In order to provide reactants to both electrodes, bipolar plates that contain flow passages are placed on both electrodes. The mass and volume overhead of the bipolar plates amounts to about 75 percent of the total mass and volume of a fuel-cell stack. Removing these bipolar plates in the micro-tubular fuel cell significantly increases the power density.

Mini-Review: Probing the limits of extremophilic life in extraterrestrial environment-simulated experiments

NASA Astrophysics Data System (ADS)

Lage, Claudia A. S.; Dalmaso, Gabriel Z. L.; Teixeira, Lia C. R. S.; Bendia, Amanda G.; Paulino-Lima, Ivan G.; Galante, Douglas; Janot-Pacheco, Eduardo; Abrevaya, Ximena C.; Azúa-Bustos, Armando; Pelizzari, Vivian H.; Rosado, Alexandre S.

2012-10-01

Astrobiology is a relatively recent scientific field that seeks to understand the origin and dynamics of life in the Universe. Several hypotheses have been proposed to explain life in the cosmic context throughout human history, but only now, technology has allowed many of them to be tested. Laboratory experiments have been able to show how chemical elements essential to life, such as carbon, nitrogen, oxygen and hydrogen combine in biologically important compounds. Interestingly, these compounds are ubiquitous. How these compounds were combined to the point of originating cells and complex organisms is still to be unveiled by science. However, our 4.5 billion years old Solar system appeared in a 10 billion years old Universe. Thus, simple cells such as micro-organisms may have had time to form in planets older than ours or in other suitable places in the Universe. One hypothesis related to the appearance of life on Earth is called panspermia, which predicts that microbial life could have been formed in the Universe billions of years ago, travelling between planets, and inseminating units of life that could have become more complex in habitable planets such as Earth. A project designed to test the viability of extremophile micro-organisms exposed to simulated extraterrestrial environments is in progress at the Carlos Chagas Filho Institute of Biophysics (UFRJ, Brazil) to test whether microbial life could withstand inhospitable environments. Radiation-resistant (known or novel ones) micro-organisms collected from extreme terrestrial environments have been exposed (at synchrotron accelerators) to intense radiation sources simulating Solar radiation, capable of emitting radiation in a few hours equivalent to many years of accumulated doses. The results obtained in these experiments reveal an interesting possibility of the existence of microbial life beyond Earth.

Epigenetic silencing of microRNA-137 enhances ASCT2 expression and tumor glutamine metabolism

PubMed Central

Dong, J; Xiao, D; Zhao, Z; Ren, P; Li, C; Hu, Y; Shi, J; Su, H; Wang, L; Liu, H; Li, B; Gao, P; Qing, G

2017-01-01

Tumor cells must activate specific transporters to meet their increased glutamine metabolic demands. Relative to other glutamine transporters, the ASC family transporter 2 (ASCT2, also called SLC1A5) is profoundly elevated in a wide spectrum of human cancers to coordinate metabolic reprogramming and malignant transformation. Understanding the molecular mechanisms whereby tumor cells frequently upregulate this transporter is therefore vital to develop potential strategies for transporter-targeted therapies. Combining in-silico algorithms with systemic experimental screening, we herein identify the tumor suppressor microRNA, miR-137, as an essential regulator that targets ASCT2 and cancer cell glutamine metabolism. Metabolic analysis shows that miR-137 derepression, similar to ASCT2 inactivation, significantly inhibits glutamine consumption and TCA cycle anaplerosis. Mechanistically, methyl-CpG-binding protein 2 (MeCP2) and DNA methyltransferases (DNMTs) cooperate to promote active methylation of the miR-137 promoter and inhibit its transcription, conversely reactivating ASCT2 expression and glutamine metabolism. Moreover, expression between miR-137 and ASCT2 is inversely correlated in tumor specimens from multiple cancer types, and ectopic ASCT2 expression markedly rescued miR-137 suppression of tumorigenesis. These findings thus elucidate a previously unreported mechanism responsible for ASCT2 deregulation in human cancers and identify ASCT2 as a critical downstream effector of miR-137, revealing a molecular link between DNA methylation, microRNA and tumor metabolism. PMID:28692032

Development of dielectrophoresis MEMS device for PC12 cell patterning to elucidate nerve-network generation

NASA Astrophysics Data System (ADS)

Nakamachi, Eiji; Koga, Hirotaka; Morita, Yusuke; Yamamoto, Koji; Sakamoto, Hidetoshi

2018-01-01

We developed a PC12 cell trapping and patterning device by combining the dielectrophoresis (DEP) methodology and the micro electro mechanical systems (MEMS) technology for time-lapse observation of morphological change of nerve network to elucidate the generation mechanism of neural network. We succeeded a neural network generation, which consisted of cell body, axon and dendrites by using tetragonal and hexagonal cell patterning. Further, the time laps observations was carried out to evaluate the axonal extension rate. The axon extended in the channel and reached to the target cell body. We found that the shorter the PC12 cell distance, the less the axonal connection time in both tetragonal and hexagonal structures. After 48 hours culture, a maximum success rate of network formation was 85% in the case of 40 μm distance tetragonal structure.

Potential proteins targeted by let-7f-5p in HeLa cells.

PubMed

Wang, Yu; Chen, Xiujuan; Zhang, Yi; Song, Jiandong

2017-07-24

MicroRNAs are a class of small, endogenous, non-coding RNAs mediating posttranscriptional gene silencing. The current authors hypothesized that let-7f-5p is likely involved in cell invasion and proliferation by regulating the expression of target genes. The current study combined let-7f-5p with iTRAQ to assess its effect on gene expression in HeLa cells. Results indicated that 164 proteins were expressed at different levels in HeLa cells overexpressing let-7f-5p and negative controls and that 172 proteins were expressed at different levels in let-7f-5p-silenced HeLa cells and negative controls. Results indicated that let-7f-5p may suppress insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in HeLa cells.

NiTi-Nb micro-trusses fabricated via extrusion-based 3D-printing of powders and transient-liquid-phase sintering.

PubMed

Taylor, Shannon L; Ibeh, Amaka J; Jakus, Adam E; Shah, Ramille N; Dunand, David C

2018-06-15

We present a novel additive manufacturing method for NiTi-Nb micro-trusses combining (i) extrusion-based 3D-printing of liquid inks containing NiTi and Nb powders, solvents, and a polymer binder into micro-trusses with 0/90° ABAB layers of parallel, ∼600 µm struts spaced 1 mm apart and (ii) subsequent heat-treatment to remove the binder and solvents, and then bond the NiTi powders using liquid phase sintering via the formation of a transient NiTi-Nb eutectic phase. We investigate the effects of Nb concentration (0, 1.5, 3.1, 6.7 at.% Nb) on the porosity, microstructure, and phase transformations of the printed NiTi-Nb micro-trusses. Micro-trusses with the highest Nb content exhibit long channels (from 3D-printing) and struts with smaller interconnected porosity (from partial sintering), resulting in overall porosities of ∼75% and low compressive stiffnesses of 1-1.6 GPa, similar to those of trabecular bone and in agreement with analytical and finite element modeling predictions. Diffusion of Nb into the NiTi particles from the bond regions results in a Ni-rich composition as the Nb replaces Ti atoms, leading to decreased martensite/austenite transformation temperatures. Adult human mesenchymal stem cells seeded on these micro-trusses showed excellent viability, proliferation, and extracellular matrix deposition over 14 days in culture. Near-equiatomic NiTi micro-trusses are attractive for biomedical applications such as stents, actuators, and bone implants because of their combination of biocompatibility, low compressive stiffness, high surface area, and shape-memory or superelasticity. Extrusion-based 3D-printing of NiTi powder-based inks into micro-trusses is feasible, but the subsequent sintering of the powders into dense struts is unachievable due to low diffusivity, large particle size, and low packing density of the NiTi powders. We present a solution, whereby Nb powders are added to the NiTi inks, thus forming during sintering a eutectic NiTi-Nb liquid phase which bonds the solid NiTi powders and improves densification of the struts. This study investigates the microstructure, porosity, phase transformation behavior, compressive stiffness, and cytocompatibility of these printed NiTi-Nb micro-trusses. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Theragnosis-based combined cancer therapy using doxorubicin-conjugated microRNA-221 molecular beacon.

PubMed

Lee, Jonghwan; Choi, Kyung-Ju; Moon, Sung Ung; Kim, Soonhag

2016-01-01

Recently, microRNA (miRNA or miR) has emerged as a new cancer biomarker because of its high expression level in various cancer types and its role in the control of tumor suppressor genes. In cancer studies, molecular imaging and treatment based on target cancer markers have been combined to facilitate simultaneous cancer diagnosis and therapy. In this study, for combined therapy with diagnosis of cancer, we developed a doxorubicin-conjugated miR-221 molecular beacon (miR-221 DOXO MB) in a single platform composed of three different nucleotides: miR-221 binding sequence, black hole quencher 1 (BHQ1), and doxorubicin binding site. Imaging of endogenous miR-221 was achieved by specific hybridization between miR-221 and the miR-221 binding site in miR-221 DOXO MB. The presence of miR-221 triggered detachment of the quencher oligo and subsequent activation of a fluorescent signal of miR-221 DOXO MB. Simultaneous cancer therapy in C6 astrocytoma cells and nude mice was achieved by inhibition of miRNA-221 function that downregulates tumor suppressor genes. The detection of miR-221 expression and inhibition of miR-221 function by miR-221 DOXO MB provide the feasibility as a cancer theragnostic probe. Furthermore, a cytotoxic effect was induced by unloading of doxorubicin intercalated into miR-221 DOXO MB inside cells. Loss of miR-221 function and cytotoxicity induced by the miR-221 DOXO MB provides combined therapeutic efficacy against cancers. This method could be used as a new theragnostic probe with enhanced therapy to detect and inhibit many cancer-related miRNAs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Calcitriol increases Dicer expression and modifies the microRNAs signature in SiHa cervical cancer cells.

PubMed

González-Duarte, Ramiro José; Cázares-Ordoñez, Verna; Romero-Córdoba, Sandra; Díaz, Lorenza; Ortíz, Víctor; Freyre-González, Julio Augusto; Hidalgo-Miranda, Alfredo; Larrea, Fernando; Avila, Euclides

2015-08-01

MicroRNAs play important roles in cancer biology. Calcitriol, the hormonal form of vitamin D3, regulates microRNAs expression in tumor cells. In the present study we asked if calcitriol would modify some of the components of the microRNA processing machinery, namely, Drosha and Dicer, in calcitriol-responsive cervical cancer cells. We found that calcitriol treatment did not affect Drosha mRNA; however, it significantly increased Dicer mRNA and protein expression in VDR-positive SiHa and HeLa cells. In VDR-negative C33-A cells, calcitriol had no effect on Dicer mRNA. We also found a vitamin D response element in Dicer promoter that interacts in vitro to vitamin D and retinoid X receptors. To explore the biological plausibility of these results, we asked if calcitriol alters the microRNA expression profile in SiHa cells. Our results revealed that calcitriol regulates the expression of a subset of microRNAs with potential regulatory functions in cancer pathways, such as miR-22, miR-296-3p, and miR-498, which exert tumor-suppressive effects. In summary, the data indicate that in SiHa cells, calcitriol stimulates the expression of Dicer possibly through the vitamin D response element located in its promoter. This may explain the calcitriol-dependent modulation of microRNAs whose target mRNAs are related to anticancer pathways, further adding to the various anticancer mechanisms of calcitriol.

Attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts.

PubMed

Moerke, Caroline; Mueller, Petra; Nebe, Barbara

2016-01-01

Cells are sensitive to their underlying micro- and nano-topography, but the complex interplay is not completely understood especially if sharp edges and ridges of stochastically modified surfaces interfere with an attached cell body. Micro-topography offers cues that evoke a large range of cell responses e.g. altered adhesion behavior and integrin expression resulting in disturbed cell functions. In this study, we analyzed why osteoblastic cells mimic the underlying geometrical micro-pillar structure (5 × 5 × 5 μm, spacing of 5 μm) with their actin cytoskeleton. Interestingly, we discovered an attempted caveolae-mediated phagocytosis of each micro-pillar beneath the cells, which was accompanied by increased intracellular reactive oxygen species (ROS) production and reduced intracellular ATP levels. This energy consuming process hampered the cells in their function as osteoblasts at the interface. The raft-dependent/caveolae-mediated phagocytic pathway is regulated by diverse cellular components including caveolin-1 (Cav-1), cholesterol, actin cytoskeleton as well as actin-binding proteins like annexin A2 (AnxA2). Our results show a new aspect of osteoblast-material interaction and give insight into how cells behave on extraordinary micro-structures. We conclude that stochastically structured implants used in orthopedic surgery should avoid any topographical heights which induce phagocytosis to prevent their successful ingrowth. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Fabrication of a co-culture micro-bioreactor device for efficient hepatic differentiation of human induced pluripotent stem cells (hiPSCs).

PubMed

Kehtari, Mousa; Zeynali, Bahman; Soleimani, Masoud; Kabiri, Mahboubeh; Seyedjafari, Ehsan

2018-04-27

Primary hepatocytes, as the gold standard cell type for in vitro models, lose their characteristic morphology and functions after few days. There is an urgent need to develop physiologically relevant models that recapitulate liver microenvironment to obtain mature hepatocyte from stem cells. We designed and fabricated a micro-bioreactor device mimicking the physiological shear stress and cell-cell interaction in liver sinusoid microenvironment. Induced pluripotent stem cells (iPSCs) were co-cultured with human umbilical vein endothelial cells (HUVECs) in the micro-bioreactor device with continuous perfusion of hepatic differentiation medium (100 μL/h). Simulation results showed that flow field inside our perfusion device was uniform and shear stress was adjusted to physiological condition (<2 dyne/cm 2 ). IPSCs-derived hepatocytes (iPSCs-Heps) that were cultured in micro-bioreactor device showed a higher level of hepatic markers compared to those in static condition. Flow cytometry and immunocytochemistry analysis revealed iPSCs cultured in the device sequentially acquired characteristics of definitive endodermal cells (SOX17 positive), hepatoblasts (AFP positive) and mature hepatocyte (ALB positive). Moreover, the albumin and urea secretion were significantly higher in micro-bioreactor device than those cultured in culture dishes during experiment. Thus, based on our results, we propose our micro-bioreactor as a beneficial device to generate mature hepatocytes for drug screening and basic research.

Natural killer cells inhibit oxaliplatin-resistant colorectal cancer by repressing WBSCR22 via upregulating microRNA-146b-5p.

PubMed

Zhao, Haiyan; Su, Wuyun; Kang, Qingmei; Xing, Ze; Lin, Xue; Wu, Zhongjun

2018-01-01

Natural killer (NK) cells have exhibited promising efficacy in inhibiting cancer growth. We aimed to explorer the effect of NK cells on oxaliplatin-resistant colorectal cancer and the underlying molecular mechanism. Oxaliplatin-resistant colorectal cancer cell lines were co-cultured with NK cells to evaluate the effect on viability, proliferation, migration and invasion in vitro . Oxaliplatin-resistant colorectal cancer cells were also co-injected with NK cells into mice to establish xenograft tumor model, to assess the in vivo effect of NK cells on tumorigenesis of the oxaliplatin-resistant colorectal cancer cells. Expression of WBSCR22 gene was assessed in the oxaliplatin-resistant colorectal cancer cells following NK cell treatment to elucidate the mechanism. NK cell treatment significantly reduces growth of oxaliplatin-resistant colorectal cancer cells both in vitro and in vivo , as well as reduced WBSCR22 expression. MicroRNAs potentially targeting WBSCR22 were analyzed, and microRNA-146b-5p was found to be significantly upregulated following NK cell treatment. MicroRNA-146b-5p directly targeted WBSCR22 mRNA 3'-UTR to inhibit its expression, which was required for NK cell-induced inhibition of oxaliplatin-resistant colorectal cancer cell lines. NK cells inhibit oxaliplatin-resistant colorectal cancer by repressing WBSCR22 via upregulating microRNA-146b-5p, both of which could serve as candidates for targeted therapy against oxaliplatin-resistant colorectal cancer.

MicroRNA-21 in laryngeal squamous cell carcinoma: Diagnostic and prognostic features.

PubMed

Erkul, Evren; Yilmaz, Ismail; Gungor, Atila; Kurt, Onuralp; Babayigit, Mustafa A

2017-02-01

We aimed to determine the microRNA-21 expression in laryngeal squamous cell carcinoma and assess the association between the disease and clinical characteristics of patients. Retrospective case-control study. A retrospective study was conducted from January 2005 to May 2011, in a tertiary hospital following tumor resection in 72 patients with laryngeal squamous cell carcinoma. We used formalin-fixed paraffin-embedded tissue samples of laryngeal squamous cell carcinomas (study group) and adjacent nontumor tissues (control group) for microRNA-21 expressions, and we successfully extracted microRNAs detectable by real-time polymerase chain reaction. All patients were evaluated separately, and the study and control groups were compared. The study group was assessed in terms of localization, smoking, alcohol consumption, lymph node staging, tumor stage, overall survival, disease-free survival, perineural, and vascular invasion. All patients were male, and the average age of patients was 64.2 ± 10.3 years. MicroRNA-21 was upregulated in laryngeal squamous cell carcinomas compared to adjacent nontumor tissues (P = .005). However, the microRNA-21 did not differ significantly according to any clinicopathological features (P > .05). MicroRNA-21 has been found to be expressed at lower levels in early stage (stages 1 and 2) compared with advanced stage (stages 3 and 4), but this was not statistically significant (P = .455). We conclude that the microRNA-21 level may play an important role in diagnosis and serve as a potential biomarker; such measurement thus has clinical applications. However, any possible prognostic associations with microRNA-21 levels should be re-evaluated in future studies on laryngeal squamous cell carcinoma samples amenable to retrospective analysis. NA Laryngoscope, 2016 127:E62-E66, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Durable and mass producible polymer surface structures with different combinations of micro-micro hierarchy

NASA Astrophysics Data System (ADS)

Jiang, Yu; Suvanto, Mika; Pakkanen, Tapani A.

2016-01-01

Extensive studies have been performed with the aim of fabricating hierarchical surface structures inspired by nature. However, synthetic hierarchical structures have to sacrifice mechanical resistance to functionality by introducing finer scaled structures. Therefore, surfaces are less durable. Surface micro-micro hierarchy has been proven to be effective in replacing micro-nano hierarchy in the sense of superhydrophobicity. However, less attention has been paid to the combined micro-micro hierarchies with surface pillars and pits incorporated together. The fabrication of this type of hierarchy may be less straightforward, with the possibility of being a complicated multi-step process. In this study, we present a simple yet mass producible fabrication method for hierarchical structures with different combinations of surface pillars and pits. The fabrication was based on only one aluminum (Al) mold with sequential mountings. The fabricated structures exhibit high mechanical durability and structural stabilities with a normal load up to 100 kg. In addition, the theoretical estimation of the wetting state shows a promising way of stabilizing a water droplet on the surface pit structures with a more stable Cassie-Baxter state.

Conceptual Layout of Wing Structure Using Topology Optimization for Morphing Micro Air Vehicles in a Perching Maneuver

DTIC Science & Technology

2012-03-22

the fraction of the design space to be filled with material (termed “volume fraction”), and any other desired design restrictions such as a ...topology problem is called a distributed parameter system because the design variables represent a field or continuum with infinite degrees of freedom... with the addition of a few solutions that were a combination of honeycomb and fiber cells. Unlike

Novel secondary assembled micro/nano porous spheres ZnCo2O4 with superior electrochemical performances as lithium ion anode material.

PubMed

Liu, Haowen; Hu, Qihong

2018-08-10

In this work, novel secondary assembled micro/nano porous spheres ZnCo 2 O 4 were firstly prepared by combining the hydrothermal method with post-synthesis calcinations. The structure and morphology of the obtained powder were characterized by x-ray powder diffraction and field emission-scanning electron microscopy. As the anode material of lithium-ion half-cells, the as-prepared ZnCo 2 O 4 delivered a very high capacity, extra cycling stability and excellent rate capability. A discharge capacity of 950 mAh g -1 with up to 99.7% retention corresponding to the second cycle at 0.1 C was achieved after 90 cycles, which was an improved cyclability over previous reports. The higher current charge-discharge and electrochemical impedance spectroscopy data indicate that the material's integrity was maintained. Therefore constructing the secondary assembled 3D micro/nano structure is an effective strategy to obtain the superior electrochemical performances.

Immunoregulatory and antioxidant performance of alpha-tocopherol and selenium on human lymphocytes.

PubMed

Lee, Chung-Yung Jetty; Wan, Jennifer Man-Fan

2002-05-01

The role of alpha-tocopherol (alpha-toco) and selenium (Se) on human lymphocyte oxidative stress and T-cells proliferation were studied by flow cytometry. We measured the hydrogen peroxide and glutathione levels in cultured human T-lymphocytes and the proliferation of their subsets: T-helper/inducer, T-suppressor/cytotoxic, and natural killer and interleukin-2 receptors upon stimulation by the mitogens phytohemaglutinin (PHA) and lipopolysaccharide (LPS). The results indicate that early stimulation by mitogens is affected by the glutathione and hydrogen peroxide status of the T-lymphocytes. The addition of 100 microM or 500 microM alpha-toco or 0.5 microM Se alone shows weak antioxidant and immunostimulant properties. When combined, an enhanced antioxidant and immunoregulatory effect was observed. The present findings indicate that alpha-toco and Se have interactive effects as oxygen radical scavengers, thus promoting human lymphocyte response to antigens. This suggests that micronutrient status is an important factor in considering when interpreting the results of in vitro assays of lymphocyte function.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-Focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feedback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was micro-fabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, micro-fabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily micro-fabricated and integrated with other polymer microfluidic structures.

System-Level Biochip for Impedance Sensing and Programmable Manipulation of Bladder Cancer Cells

PubMed Central

Chuang, Cheng-Hsin; Huang, Yao-Wei; Wu, Yao-Tung

2011-01-01

This paper develops a dielectrophoretic (DEP) chip with multi-layer electrodes and a micro-cavity array for programmable manipulations of cells and impedance measurement. The DEP chip consists of an ITO top electrode, flow chamber, middle electrode on an SU-8 surface, micro-cavity arrays of SU-8 and distributed electrodes at the bottom of the micro-cavity. Impedance sensing of single cells could be performed as follows: firstly, cells were trapped in a micro-cavity array by negative DEP force provided by top and middle electrodes; then, the impedance measurement for discrimination of different stage of bladder cancer cells was accomplished by the middle and bottom electrodes. After impedance sensing, the individual releasing of trapped cells was achieved by negative DEP force using the top and bottom electrodes in order to collect the identified cells once more. Both cell manipulations and impedance measurement had been integrated within a system controlled by a PC-based LabVIEW program. In the experiments, two different stages of bladder cancer cell lines (grade III: T24 and grade II: TSGH8301) were utilized for the demonstration of programmable manipulation and impedance sensing; as the results show, the lower-grade bladder cancer cells (TSGH8301) possess higher impedance than the higher-grade ones (T24). In general, the multi-step manipulations of cells can be easily programmed by controlling the electrical signal in our design, which provides an excellent platform technology for lab-on-a-chip (LOC) or a micro-total-analysis-system (Micro TAS). PMID:22346685

MicroRNA regulation of endothelial homeostasis and commitment-implications for vascular regeneration strategies using stem cell therapies.

PubMed

Scott, Elizabeth; Loya, Komal; Mountford, Joanne; Milligan, Graeme; Baker, Andrew H

2013-09-01

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Specific and Novel microRNAs Are Regulated as Response to Fungal Infection in Human Dendritic Cells.

PubMed

Dix, Andreas; Czakai, Kristin; Leonhardt, Ines; Schäferhoff, Karin; Bonin, Michael; Guthke, Reinhard; Einsele, Hermann; Kurzai, Oliver; Löffler, Jürgen; Linde, Jörg

2017-01-01

Within the last two decades, the incidence of invasive fungal infections has been significantly increased. They are characterized by high mortality rates and are often caused by Candida albicans and Aspergillus fumigatus . The increasing number of infections underlines the necessity for additional anti-fungal therapies, which require extended knowledge of gene regulations during fungal infection. MicroRNAs are regulators of important cellular processes, including the immune response. By analyzing their regulation and impact on target genes, novel therapeutic and diagnostic approaches may be developed. Here, we examine the role of microRNAs in human dendritic cells during fungal infection. Dendritic cells represent the bridge between the innate and the adaptive immune systems. Therefore, analysis of gene regulation of dendritic cells is of particular significance. By applying next-generation sequencing of small RNAs, we quantify microRNA expression in monocyte-derived dendritic cells after 6 and 12 h of infection with C. albicans and A. fumigatus as well as treatment with lipopolysaccharides (LPS). We identified 26 microRNAs that are differentially regulated after infection by the fungi or LPS. Three and five of them are specific for fungal infections after 6 and 12 h, respectively. We further validated interactions of miR-132-5p and miR-212-5p with immunological relevant target genes, such as FKBP1B, KLF4 , and SPN , on both RNA and protein level. Our results indicate that these microRNAs fine-tune the expression of immune-related target genes during fungal infection. Beyond that, we identified previously undiscovered microRNAs. We validated three novel microRNAs via qRT-PCR. A comparison with known microRNAs revealed possible relations with the miR-378 family and miR-1260a/b for two of them, while the third one features a unique sequence with no resemblance to known microRNAs. In summary, this study analyzes the effect of known microRNAs in dendritic cells during fungal infections and proposes novel microRNAs that could be experimentally verified.

Multilayered membranes with tuned well arrays to be used as regenerative patches.

PubMed

Martins, Nádia I; Sousa, Maria P; Custódio, Catarina A; Pinto, Vânia C; Sousa, Paulo J; Minas, Graça; Cleymand, Franck; Mano, João F

2017-07-15

Membranes have been explored as patches in tissue repair and regeneration, most of them presenting a flat geometry or a patterned texture at the nano/micrometer scale. Herein, a new concept of a flexible membrane featuring well arrays forming pore-like environments to accommodate cell culture is proposed. The processing of such membranes using polysaccharides is based on the production of multilayers using the layer-by-layer methodology over a patterned PDMS substrate. The detached multilayered membrane exhibits a layer of open pores at one side and a total thickness of 38±2.2µm. The photolithography technology used to produce the molds allows obtaining wells on the final membranes with a tuned shape and micro-scale precision. The influence of post-processing procedures over chitosan/alginate films with 100 double layers, including crosslinking with genipin or fibronectin immobilization, on the adhesion and proliferation of human osteoblast-like cells is also investigated. The results suggest that the presence of patterned wells affects positively cell adhesion, morphology and proliferation. In particular, it is seen that cells colonized preferentially the well regions. The geometrical features with micro to sub-millimeter patterned wells, together with the nano-scale organization of the polymeric components along the thickness of the film will allow to engineer highly versatile multilayered membranes exhibiting a pore-like microstructure in just one of the sides, that could be adaptable in the regeneration of multiple tissues. Flexible multilayered membranes containing multiple micro-reservoirs are found as potential regenerative patches. Layer-by-layer (LbL) methodology over a featured PDMS substrate is used to produce patterned membranes, composed only by natural-based polymers, that can be easily detached from the PDMS substrate. The combination of nano-scale control of the polymeric organization along the thickness of the chitosan/alginate (CHT/ALG) membranes, provided by LbL, together with the geometrical micro-scale features of the patterned membranes offers a uniqueness system that allows cells to colonize 3-dimensionally. This study provides a promising strategy to control cellular spatial organization that can face the region of the tissue to regenerate. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Anti-proliferative and differentiation-inducing activities of the green tea catechin epigallocatechin-3-gallate (EGCG) on the human eosinophilic leukemia EoL-1 cell line.

PubMed

Lung, H L; Ip, W K; Wong, C K; Mak, N K; Chen, Z Y; Leung, K N

2002-12-06

A novel approach for the treatment of leukemia is the differentiation therapy in which immature leukemia cells are induced to attain a mature phenotype when exposed to differentiation inducers, either alone or in combinations with other chemotherapeutic or chemopreventive drugs. Over the past decade, numerous studies indicated that green tea catechins (GTC) could suppress the growth and induce apoptosis on a number of human cancer cell lines. However, the differentiation-inducing activity of GTC on human tumors remains poorly understood. In the present study, the effect of the major GTC epigallocatechin-3-gallate (EGCG) on the proliferation and differentiation of a human eosinophilc leukemic cell line, EoL-1, was examined. Our results showed that EGCG suppressed the proliferation of the EoL-1 cells in a dose-dependent manner, with an estimated IC(50) value of 31.5 microM. On the other hand, EGCG at a concentration of 40 microM could trigger the EoL-1 cells to undergo morphological differentiation into mature eosinophil-like cells. Using RT-PCR and flow cytometry, it was found that EGCG upregulated the gene and protein expression of two eosinophil-specific granule proteins, the major basic protein (MBP) and eosinophil peroxidase (EPO), in EoL-1 cells. Taken together, our findings suggest that EGCG can exhibit anti-leukemic activity on a human eosinophilic cell line EoL-1 by suppressing the proliferation and by inducing the differentiation of the leukemia cells.

Impaired expression of DICER and some microRNAs in HBZ expressing cells from acute adult T-cell leukemia patients

PubMed Central

Gazon, Hélène; Belrose, Gildas; Terol, Marie; Meniane, Jean-Come; Mesnard, Jean-Michel; Césaire, Raymond; Peloponese, Jean-Marie

2016-01-01

Global dysregulation of microRNAs (miRNAs), a class of non-coding RNAs that regulate genes expression, is a common feature of human tumors. Profiling of cellular miRNAs on Adult T cell Leukemia (ATL) cells by Yamagishi et al. showed a strong decrease in expression for 96.7% of cellular miRNAs in ATL cells. However, the mechanisms that regulate the expression of miRNAs in ATL cells are still largely unknown. In this study, we compared the expression of 12 miRs previously described for being overexpress by Tax and the expression of several key components of the miRNAs biogenesis pathways in different HBZ expressing cell lines as well as in primary CD4 (+) cells from acute ATL patients. We showed that the expression of miRNAs and Dicer1 were downregulated in cells lines expressing HBZ as well as in fresh CD4 (+) cells from acute ATL patients. Using qRT-PCR, western blotting analysis and Chromatin Immunoprecipitation, we showed that dicer transcription was regulated by c-Jun and JunD, two AP-1 transcription factors. We also demonstrated that HBZ affects the expression of Dicer by removing JunD from the proximal promoter. Furthermore, we showed that at therapeutic concentration of 1mM, Valproate (VPA) an HDAC inhibitors often used in cancer treatment, rescue Dicer expression and miRNAs maturation. These results might offer a rationale for clinical studies of new combined therapy in an effort to improve the outcome of patients with acute ATL. PMID:26849145

Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsutsui, Motomu; Hasegawa, Hitoki; Adachi, Koichi

Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have notmore » responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.« less

Protein and cell micropatterning and its integration with micro/nanoparticles assembly.

PubMed

Yap, F L; Zhang, Y

2007-01-15

Micropatterning of proteins and cells has become very popular over the past decade due to its importance in the development of biosensors, microarrays, tissue engineering and cellular studies. This article reviews the techniques developed for protein and cell micropatterning and its biomedical applications. The prospect of integrating micro and nanoparticles with protein and cell micropatterning is discussed. The micro/nanoparticles are assembled into patterns and form the substrate for proteins and cell attachment. The assembled particles create a micro or nanotopography, depending on the size of the particles employed. The nonplanar structure can increase the surface area for biomolecules attachment and therefore enhance the sensitivity for detection in biosensors. Furthermore, a nanostructured substrate can influence the conformation and functionality of protein attached to it, while cellular response in terms of morphology, adhesion, proliferation, differentiation, etc. can be affected by a surface expressing micro or nanoscale structures. Proteins and cells tend to lose their normal functions upon attachment to substrate. By recognizing the types of topography that are favourable for preserving proteins and cell behaviour, and integrating it with micropattering will lead to the development of functional protein and cell patterns.

Basal cell carcinoma pathogenesis and therapy involving hedgehog signaling and beyond.

PubMed

Bakshi, Anshika; Chaudhary, Sandeep C; Rana, Mehtab; Elmets, Craig A; Athar, Mohammad

2017-12-01

Basal cell carcinoma (BCC) of the skin is driven by aberrant hedgehog signaling. Thus blocking this signaling pathway by small molecules such as vismodegib inhibits tumor growth. Primary cilium in the epidermal cells plays an integral role in the processing of hedgehog signaling-related proteins. Recent genomic studies point to the involvement of additional genetic mutations that might be associated with the development of BCCs, suggesting significance of other signaling pathways, such as WNT, NOTCH, mTOR, and Hippo, aside from hedgehog in the pathogenesis of this human neoplasm. Some of these pathways could be regulated by noncoding microRNA. Altered microRNA expression profile is recognized with the progression of these lesions. Stopping treatment with Smoothened (SMO) inhibitors often leads to tumor reoccurrence in the patients with basal cell nevus syndrome, who develop 10-100 of BCCs. In addition, the initial effectiveness of these SMO inhibitors is impaired due to the onset of mutations in the drug-binding domain of SMO. These data point to a need to develop strategies to overcome tumor recurrence and resistance and to enhance efficacy by developing novel single agent-based or multiple agents-based combinatorial approaches. Immunotherapy and photodynamic therapy could be additional successful approaches particularly if developed in combination with chemotherapy for inoperable and metastatic BCCs. © 2017 Wiley Periodicals, Inc.

Basal cell carcinoma pathogenesis and therapy involving hedgehog signaling and beyond

PubMed Central

Bakshi, Anshika; Chaudhary, Sandeep C.; Rana, Mehtab; Elmets, Craig A.; Athar, Mohammad

2018-01-01

Basal cell carcinoma (BCC) of the skin is driven by aberrant hedgehog signaling. Thus blocking this signaling pathway by small molecules such as vismodegib inhibits tumor growth. Primary cilium in the epidermal cells plays an integral role in the processing of hedgehog signaling-related proteins. Recent genomic studies point to the involvement of additional genetic mutations that might be associated with the development of BCCs, suggesting significance of other signaling pathways, such as WNT, NOTCH, mTOR, and Hippo, aside from hedgehog in the pathogenesis of this human neoplasm. Some of these pathways could be regulated by noncoding microRNA. Altered microRNA expression profile is recognized with the progression of these lesions. Stopping treatment with Smoothened (SMO) inhibitors often leads to tumor reoccurrence in the patients with basal cell nevus syndrome, who develop 10–100 of BCCs. In addition, the initial effectiveness of these SMO inhibitors is impaired due to the onset of mutations in the drug-binding domain of SMO. These data point to a need to develop strategies to overcome tumor recurrence and resistance and to enhance efficacy by developing novel single agent-based or multiple agents-based combinatorial approaches. Immunotherapy and photodynamic therapy could be additional successful approaches particularly if developed in combination with chemotherapy for inoperable and metastatic BCCs. PMID:28574612

Polysilicon-chromium-gold intracellular chips for multi-functional biomedical applications

NASA Astrophysics Data System (ADS)

Patiño, Tania; Soriano, Jorge; Amirthalingam, Ezhil; Durán, Sara; González-Campo, Arántzazu; Duch, Marta; Ibáñez, Elena; Barrios, Leonardo; Plaza, Jose Antonio; Pérez-García, Lluïsa; Nogués, Carme

2016-04-01

The development of micro- and nanosystems for their use in biomedicine is a continuously growing field. One of the major goals of such platforms is to combine multiple functions in a single entity. However, achieving the design of an efficient and safe micro- or nanoplatform has shown to be strongly influenced by its interaction with the biological systems, where particle features or cell types play a critical role. In this work, the feasibility of using multi-material pSi-Cr-Au intracellular chips (MMICCs) for multifunctional applications by characterizing their interactions with two different cell lines, one tumorigenic and one non-tumorigenic, in terms of biocompatibility, internalization and intracellular fate, has been explored. Moreover, the impact of MMICCs on the induction of an inflammatory response has been assessed by evaluating TNFα, IL1b, IL6, and IL10 human inflammatory cytokines secretion by macrophages. Results show that MMICCs are biocompatible and their internalization efficiency is strongly dependent on the cell type. Finally as a proof-of-concept, MMICCs have been dually functionalized with transferrin and pHrodo™ Red, SE to target cancer cells and detect intracellular pH, respectively. In conclusion, MMICCs can be used as multi-functional devices due to their high biocompatibility, non-inflammatory properties and the ability of developing multiple functions.

Polysilicon-chromium-gold intracellular chips for multi-functional biomedical applications.

PubMed

Patiño, Tania; Soriano, Jorge; Amirthalingam, Ezhil; Durán, Sara; González-Campo, Arántzazu; Duch, Marta; Ibáñez, Elena; Barrios, Leonardo; Plaza, Jose Antonio; Pérez-García, Lluïsa; Nogués, Carme

2016-04-28

The development of micro- and nanosystems for their use in biomedicine is a continuously growing field. One of the major goals of such platforms is to combine multiple functions in a single entity. However, achieving the design of an efficient and safe micro- or nanoplatform has shown to be strongly influenced by its interaction with the biological systems, where particle features or cell types play a critical role. In this work, the feasibility of using multi-material pSi-Cr-Au intracellular chips (MMICCs) for multifunctional applications by characterizing their interactions with two different cell lines, one tumorigenic and one non-tumorigenic, in terms of biocompatibility, internalization and intracellular fate, has been explored. Moreover, the impact of MMICCs on the induction of an inflammatory response has been assessed by evaluating TNFα, IL1b, IL6, and IL10 human inflammatory cytokines secretion by macrophages. Results show that MMICCs are biocompatible and their internalization efficiency is strongly dependent on the cell type. Finally as a proof-of-concept, MMICCs have been dually functionalized with transferrin and pHrodo™ Red, SE to target cancer cells and detect intracellular pH, respectively. In conclusion, MMICCs can be used as multi-functional devices due to their high biocompatibility, non-inflammatory properties and the ability of developing multiple functions.

Hydrogel microfluidics for the patterning of pluripotent stem cells

NASA Astrophysics Data System (ADS)

Cosson, S.; Lutolf, M. P.

2014-03-01

Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate.

Development of ultrasonic electrostatic microjets for distributed propulsion and microflight

NASA Astrophysics Data System (ADS)

Amirparviz, Babak

This dissertation details the first attempt to design and fabricate a distributed micro propulsion system based on acoustic streaming. A novel micro propulsion method is suggested by combining Helmholtz resonance, acoustic streaming and flow entrainment and thrust augmentation. In this method, oscillatory motion of an electrostatically actuated diaphragm creates a high frequency acoustic field inside the cavity of a Helmholtz resonator. The initial fluid motion velocity is amplified by the Helmholtz resonator structure and creates a jet flow at the exit nozzle. Acoustic streaming is the phenomenon responsible for primary jet stream creation. Primary jets produced by a few resonators can be combined in an ejector configuration to induce flow entrainment and thrust augmentation. Basic governing equations for the electrostatic actuator, deformation of the diaphragm and the fluid flow inside the resonator are derived. These equations are linearized and used to derive an equivalent electrical circuit model for the operation of the device. Numerical solution of the governing equations and simulation of the circuit model are used to predict the performance of the experimental systems. Thrust values as high as 30.3muN are expected per resonator. A micro machined electrostatically-driven high frequency Helmholtz resonator prototype is designed and fabricated. A new micro fabrication technique is developed for bulk micromachining and in particular fabrication of the resonator. Geometric stops for wet anisotropic etching of silicon are introduced for the fist time for structure formation. Arrays of high frequency (>60kHz) micro Helmholtz resonators are fabricated. In one sample more than 1000 resonators cover the surface of a four-inch silicon wafer and in effect convert it to a distributed propulsion system. A high yield (>85%) micro fabrication process is presented for realization of this propulsion system taking advantage of newly developed deep glass micromachining and lithography on thin (15mum) silicon methods. Extensive test and characterization are performed on the micro jets using current frequency component analysis, laser interferometry, acoustic measurements, hot-wire anemometers, video particle imaging and load cells. The occurrence of acoustic streaming at jet nozzles is verified and flow velocities exceeding 1m/s are measured at the 15mum x 330mum jet exit nozzle.

Identification of let-7a-2-3p or/and miR-188-5p as Prognostic Biomarkers in Cytogenetically Normal Acute Myeloid Leukemia

PubMed Central

Jinlong, Shi; Lin, Fu; Yonghui, Li; Li, Yu; Weidong, Wang

2015-01-01

Cytogenetically normal acute myeloid leukemia (CN-AML) is the largest and most heterogeneous AML subgroup. It lacks sensitive and specific biomarkers. Emerging evidences have suggested that microRNAs are involved in the pathogenesis of various leukemias. This paper evaluated the association between microRNA expression and prognostic outcome for CN-AML, based on the RNA/microRNA sequencing data of CN-AML patients. High let-7a-2-3p expression and low miR-188-5p expression were identified to be significantly associated with longer overall survival (OS) and event free survival (EFS) for CN-AML, independently or in a combined way. Their prognostic values were further confirmed in European Leukemia Net (ELN) genetic categories. Also, in multivariable analysis with other known risk factors, high let-7a-2-3p and low miR-188-5p expression remained to be associated with longer OS and EFS. In addition, the prognostic value of these two microRNAs was confirmed in patients who received hematopoietic stem cell transplantation (HSCT). To gain more biological insights of the underlying mechanisms, we derived the genome-wide differential gene/microRNA signatures associated with the expression of let-7a-2-3p and miR-188-5p. Several common microRNA signatures indicating favorable outcome in previous studies were up-regulated in both high let-7a-2-3p expressers and low miR-188-5p expressers, including miR-135a, miR-335 and miR-125b and all members of miR-181 family. Additionally, common up-regulated genes included FOSB, IGJ, SNORD50A and ZNF502, and FOSB was a known favorable signature in AML. These common signatures further confirmed the underlying common mechanisms for these two microRNAs value as favorable prognostic biomarkers. We concluded that high let-7a-2-3p and low miR-188-5p expression could be potentially used as favorably prognostic biomarkers independently or in a combined way in CN-AML patients, whether they received HSCT or not. PMID:25646775

Identification of let-7a-2-3p or/and miR-188-5p as prognostic biomarkers in cytogenetically normal acute myeloid leukemia.

PubMed

Jinlong, Shi; Lin, Fu; Yonghui, Li; Li, Yu; Weidong, Wang

2015-01-01

Cytogenetically normal acute myeloid leukemia (CN-AML) is the largest and most heterogeneous AML subgroup. It lacks sensitive and specific biomarkers. Emerging evidences have suggested that microRNAs are involved in the pathogenesis of various leukemias. This paper evaluated the association between microRNA expression and prognostic outcome for CN-AML, based on the RNA/microRNA sequencing data of CN-AML patients. High let-7a-2-3p expression and low miR-188-5p expression were identified to be significantly associated with longer overall survival (OS) and event free survival (EFS) for CN-AML, independently or in a combined way. Their prognostic values were further confirmed in European Leukemia Net (ELN) genetic categories. Also, in multivariable analysis with other known risk factors, high let-7a-2-3p and low miR-188-5p expression remained to be associated with longer OS and EFS. In addition, the prognostic value of these two microRNAs was confirmed in patients who received hematopoietic stem cell transplantation (HSCT). To gain more biological insights of the underlying mechanisms, we derived the genome-wide differential gene/microRNA signatures associated with the expression of let-7a-2-3p and miR-188-5p. Several common microRNA signatures indicating favorable outcome in previous studies were up-regulated in both high let-7a-2-3p expressers and low miR-188-5p expressers, including miR-135a, miR-335 and miR-125b and all members of miR-181 family. Additionally, common up-regulated genes included FOSB, IGJ, SNORD50A and ZNF502, and FOSB was a known favorable signature in AML. These common signatures further confirmed the underlying common mechanisms for these two microRNAs value as favorable prognostic biomarkers. We concluded that high let-7a-2-3p and low miR-188-5p expression could be potentially used as favorably prognostic biomarkers independently or in a combined way in CN-AML patients, whether they received HSCT or not.

Infrared micro-spectroscopic studies of epithelial cells

PubMed Central

Romeo, Melissa; Mohlenhoff, Brian; Jennings, Michael; Diem, Max

2009-01-01

We report results from a study of human and canine mucosal cells, investigated by infrared micro-spectroscopy, and analyzed by methods of multivariate statistics. We demonstrate that the infrared spectra of individual cells are sensitive to the stage of maturation, and that a distinction between healthy and diseased cells will be possible. Since this report is written for an audience not familiar with infrared micro-spectroscopy, a short introduction into this field is presented along with a summary of principal component analysis. PMID:16797481

[Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model].

PubMed

Yuan, Wei; Zheng, Jun; Qian, Jinyu; Zhou, Xiaoxiao; Wang, Minghui; Wang, Xiuhui

2017-07-01

To observe the effect of stromal vascular fraction cells (SVFs) from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) in promoting the lumbar fusion in rat model. SVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β-tricalcium phosphate (β-TCP) by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix (DBX) scaffold+PBS, DBX scaffold+rhBMP-2/β-TCP sustained release carrier, DBX scaffold+SVFs, and DBX scaffold+rhBMP-2/β-TCP sustained release carrier+SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density (BMD) and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson's trichrome staining, osteocalcin (OCN) was detected by immunohistochemical staining. The cumulative release amount of rhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation (100%, 8/8), followed by group B (75%, 6/8), group C (37.5%, 3/8), and group A (12.5%, 1/8). Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C ( P <0.05), and in group B than groups A and C ( P <0.05). HE staining, Masson's trichrome staining, and immunohistochemistry staining for OCN staining exhibited a large number of cartilage cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and there was no effective new bone formation in groups A and C. The combination of sustained release of rhBMP-2 and freshly SVFs can significantly promote spinal fusion in rat model, providing a theoretical basis for further clinical applications.

Generation of tooth-periodontium complex structures using high-odontogenic potential dental epithelium derived from mouse embryonic stem cells.

PubMed

Zhang, Yancong; Li, Yongliang; Shi, Ruirui; Zhang, Siqi; Liu, Hao; Zheng, Yunfei; Li, Yan; Cai, Jinglei; Pei, Duanqing; Wei, Shicheng

2017-06-08

A number of studies have shown that tooth-like structures can be regenerated using induced pluripotent stem cells and mouse embryonic stem (mES) cells. However, few studies have reported the regeneration of tooth-periodontium complex structures, which are more suitable for clinical tooth transplantation. We established an optimized approach to induce high-odontogenic potential dental epithelium derived from mES cells by temporally controlling bone morphogenic protein 4 (BMP4) function and regenerated tooth-periodontium complex structures in vivo. First, immunofluorescence and quantitative reverse transcription-polymerase chain reaction were used to identify the watershed of skin and the oral ectoderm. LDN193189 was then used to inhibit the BMP4 receptor around the watershed, followed by the addition of exogenous BMP4 to promote BMP4 function. The generated dental epithelium was confirmed by western blot analysis and immunofluorescence. The generated epithelium was ultimately combined with embryonic day 14.5 mouse mesenchyme and transplanted into the renal capsules of nude mice. After 4 weeks, the tooth-periodontium complex structure was examined by micro-computed tomography (CT) and hematoxylin and eosin (H&E) staining. Our study found that the turning point of oral ectoderm differentiation occurred around day 3 after the embryoid body was transferred to a common culture plate. Ameloblastin-positive dental epithelial cells were detected following the temporal regulation of BMP4. Tooth-periodontium complex structures, which included teeth, a periodontal membrane, and alveolar bone, were formed when this epithelium was combined with mouse dental mesenchyme and transplanted into the renal capsules of nude mice. Micro-CT and H&E staining revealed that the generated tooth-periodontium complex structures shared a similar histological structure with normal mouse teeth. An optimized induction method was established to promote the differentiation of mES cells into dental epithelium by temporally controlling the function of BMP4. A novel tooth-periodontium complex structure was generated using the epithelium.

Flow velocity-driven differentiation of human mesenchymal stromal cells in silk fibroin scaffolds: A combined experimental and computational approach.

PubMed

Vetsch, Jolanda Rita; Betts, Duncan Colin; Müller, Ralph; Hofmann, Sandra

2017-01-01

Mechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (vlow = 0.001m/s and vhigh = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at vlow compared to vhigh and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at vhigh whereas the amount of DNA was increased at vlow. The biological response at vlow might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (vlow) or bone remodeling (vhigh). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading.

Genotoxicity of mercury used in chromosome aberration tests.

PubMed

Akiyama, M; Oshima, H; Nakamura, M

2001-01-01

The purpose of this study was to investigate the genotoxic effects of Hg released from dental amalgams. The chromosome aberration test was conducted using original extracts and their diluted solutions of conventional type amalgam and high copper amalgam. The concentrations of Hg, Cu and Ag in the original extract of high copper amalgam were 17.64, 7.97 and 43.90 microM, respectively. Those in the original extract of conventional type amalgam were 20.63, 7.87 and 14.79 microM, respectively. 10 and 30 microM Hg(2+) were also used for comparison. The frequency of chromosome aberrations was below 5% with 0 microM Hg(2+) and with a triple dilution of high copper amalgam extract, containing 5.88 microM Hg, 14.63 microM Cu and 2.65 microM Ag. However, 9.5% of the cells showed chromosome aberrations with a quadruple dilution of conventional type amalgam, containing 5.15 microM Hg, 3.69 microM Cu and 1.96 microM Ag. The amount of Hg in the quadruple dilution of conventional type amalgam was less than that in the triple dilution of high copper amalgam extract and 10 microM Hg(2+). A concentration of 30 microM Hg(2+) caused 34.5% of the cells to show chromosome aberrations while with a two-thirds dilution of high copper amalgam extract, containing 11.76 microM Hg, 29.26 microM Cu and 5.31 microM Ag, 58.5% of the cells showed chromosome aberrations. A two-thirds dilution of high copper amalgam extract induced more chromosome aberrations than 30 microM Hg(2+), although the amount of Hg was less than 30 microM Hg(2+). A triple dilution of conventional type amalgam extract, original extracts of conventional type amalgam and high copper amalgam and 100 microM Hg(2+) were induced few metaphases. It was revealed that the conventional type amalgam induced chromosome aberrations with quadruple dilution where cell viability was about 80% and that the high copper amalgam induced a high level of chromosome aberrations with the two-thirds dilution. The effects of low level Hg on humans are not clear.

Bio-metals imaging and speciation in cells using proton and synchrotron radiation X-ray microspectroscopy

PubMed Central

Ortega, Richard; Devès, Guillaume; Carmona, Asunción

2009-01-01

The direct detection of biologically relevant metals in single cells and of their speciation is a challenging task that requires sophisticated analytical developments. The aim of this article is to present the recent achievements in the field of cellular chemical element imaging, and direct speciation analysis, using proton and synchrotron radiation X-ray micro- and nano-analysis. The recent improvements in focusing optics for MeV-accelerated particles and keV X-rays allow application to chemical element analysis in subcellular compartments. The imaging and quantification of trace elements in single cells can be obtained using particle-induced X-ray emission (PIXE). The combination of PIXE with backscattering spectrometry and scanning transmission ion microscopy provides a high accuracy in elemental quantification of cellular organelles. On the other hand, synchrotron radiation X-ray fluorescence provides chemical element imaging with less than 100 nm spatial resolution. Moreover, synchrotron radiation offers the unique capability of spatially resolved chemical speciation using micro-X-ray absorption spectroscopy. The potential of these methods in biomedical investigations will be illustrated with examples of application in the fields of cellular toxicology, and pharmacology, bio-metals and metal-based nano-particles. PMID:19605403

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells.

PubMed

Mishima, Masayuki; Tanaka, Kenji; Takeiri, Akira; Harada, Asako; Kubo, Chiyomi; Sone, Sachiko; Nishimura, Yoshikazu; Tachibana, Yukako; Okazaki, Makoto

2008-08-25

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2 microM, 2.4 microM, and 4.8 microM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8 microM, 3.6 microM, and 5.4 microM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.

A micromachined carbon nanotube film cantilever-based energy cell

NASA Astrophysics Data System (ADS)

Gong, Zhongcheng; He, Yuan; Tseng, Yi-Hsuan; O'Neal, Chad; Que, Long

2012-08-01

This paper reports a new type of energy cell based on micromachined carbon nanotube film (CNF)-lead zirconate titanate cantilevers that is fabricated on silicon substrates. Measurements found that this type of micro-energy cell generates both AC voltages due to the self-reciprocation of the microcantilevers and DC voltages due to the thermoelectric effect upon exposure to light and thermal radiation, resulting from the unique optical and thermal properties of the CNF. Typically the measured power density of the micro-energy cell can be from 4 to 300 μW cm-2 when it is exposed to sunlight under different operational conditions. It is anticipated that hundreds of integrated micro-energy cells can generate power in the range of milliwatts, paving the way for the construction of self-powered micro- or nanosystems.

Effect of Low-Intensity Pulsed Ultrasound after Mesenchymal Stromal Cell Injection to Treat Osteochondral Defects: An In Vivo Study.

PubMed

Yamaguchi, Shoki; Aoyama, Tomoki; Ito, Akira; Nagai, Momoko; Iijima, Hirotaka; Tajino, Junichi; Zhang, Xiangkai; Wataru, Kiyan; Kuroki, Hiroshi

2016-12-01

We investigated the effect of low-intensity pulsed ultrasound (LIPUS) treatment combined with mesenchymal stromal cell (MSC) injection for cartilage repair and subchondral bone reconstitution for treatment of osteochondral defects. An osteochondral defect was created on both femur grooves of Wistar rats. Four weeks later, bone marrow MSCs were injected into the right knee joint. The rats were divided into two intervention groups: without or with LIPUS irradiation. Cartilage repair was evaluated histologically based on the Wakitani cartilage repair score. Subchondral bone reconstitution was evaluated as bone volume (BV)/tissue volume (TV) by micro-computed tomography analysis. MSC injection improved the cartilage repair score, and LIPUS irradiation improved BV/TV. Combination treatment promoted both cartilage repair and BV/TV improvement. Thus, MSC injection combined with LIPUS irradiation is more effective than either treatment alone in promoting concurrent cartilage repair and subchondral reconstitution. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

DNA methylation, microRNAs, and their crosstalk as potential biomarkers in hepatocellular carcinoma

PubMed Central

Anwar, Sumadi Lukman; Lehmann, Ulrich

2014-01-01

Epigenetic alterations have been identified as a major characteristic in human cancers. Advances in the field of epigenetics have contributed significantly in refining our knowledge of molecular mechanisms underlying malignant transformation. DNA methylation and microRNA expression are epigenetic mechanisms that are widely altered in human cancers including hepatocellular carcinoma (HCC), the third leading cause of cancer related mortality worldwide. Both DNA methylation and microRNA expression patterns are regulated in developmental stage specific-, cell type specific- and tissue-specific manner. The aberrations are inferred in the maintenance of cancer stem cells and in clonal cell evolution during carcinogenesis. The availability of genome-wide technologies for DNA methylation and microRNA profiling has revolutionized the field of epigenetics and led to the discovery of a number of epigenetically silenced microRNAs in cancerous cells and primary tissues. Dysregulation of these microRNAs affects several key signalling pathways in hepatocarcinogenesis suggesting that modulation of DNA methylation and/or microRNA expression can serve as new therapeutic targets for HCC. Accumulative evidence shows that aberrant DNA methylation of certain microRNA genes is an event specifically found in HCC which correlates with unfavorable outcomes. Therefore, it can potentially serve as a biomarker for detection as well as for prognosis, monitoring and predicting therapeutic responses in HCC. PMID:24976726

Gap junctions modulate glioma invasion by direct transfer of microRNA.

PubMed

Hong, Xiaoting; Sin, Wun Chey; Harris, Andrew L; Naus, Christian C

2015-06-20

The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity.

Gap junctions modulate glioma invasion by direct transfer of microRNA

PubMed Central

Hong, Xiaoting; Sin, Wun Chey; Harris, Andrew L.; Naus, Christian C.

2015-01-01

The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity. PMID:25978028

Expression of Herpes Simplex Virus 1-Encoded MicroRNAs in Human Trigeminal Ganglia and Their Relation to Local T-Cell Infiltrates ▿

PubMed Central

Held, Kathrin; Junker, Andreas; Dornmair, Klaus; Meinl, Edgar; Sinicina, Inga; Brandt, Thomas; Theil, Diethilde; Derfuss, Tobias

2011-01-01

Herpes simplex type 1 (HSV-1) is a neurotropic virus which establishes lifelong latency in human trigeminal ganglia (TG). Currently, two nonexclusive control mechanisms of HSV-1 latency are discussed: antiviral CD8+ T cells and viral microRNAs (miRNAs) encoded by the latency associated transcript (LAT). We investigate here to what extent these mechanisms may contribute to the maintenance of HSV-1 latency. We show that only a small proportion of LAT+ neurons is surrounded by T cells in human TG. This indicates that viral latency in human TG might be controlled by other mechanisms such as viral miRNAs. Therefore, we assessed TG sections for the presence of HSV-1 miRNA, DNA, and mRNA by combining LAT in situ hybridization, T-cell immunohistochemistry, and single cell analysis of laser-microdissected sensory neurons. Quantitative reverse transcription-PCR (RT-PCR) revealed that LAT+ neurons with or without surrounding T cells were always positive for HSV-1 miRNAs and DNA. Furthermore, ICP0 mRNA could rarely be detected only in LAT+ neurons, as analyzed by single-cell RT-PCR. In contrast, in LAT− neurons that were surrounded by T cells, neither miRNAs nor the DNA of HSV-1, HSV-2, or varicella-zoster virus could be detected. These data indicate that the majority of LAT+ neurons is not directly controlled by T cells. However, miRNA expression in every latently infected neuron would provide an additional checkpoint before viral replication is initiated. PMID:21795359

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

PubMed Central

Fruhwirth, Gilbert O.; Ameer-Beg, Simon; Cook, Richard; Watson, Timothy; Ng, Tony; Festy, Frederic

2010-01-01

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications. PMID:20588974

Human microRNA-1245 down-regulates the NKG2D receptor in natural killer cells and impairs NKG2D-mediated functions

PubMed Central

Espinoza, J. Luis; Takami, Akiyoshi; Yoshioka, Katsuji; Nakata, Katsuya; Sato, Tokiharu; Kasahara, Yoshihito; Nakao, Shinji

2012-01-01

Background NKG2D is an activating receptor expressed by natural killer and T cells, which have crucial functions in tumor and microbial immunosurveillance. Several cytokines have been identified as modulators of NKG2D receptor expression. However, little is known about NKG2D gene regulation. In this study, we found that microRNA 1245 attenuated the expression of NKG2D in natural killer cells. Design and Methods We investigated the potential interactions between the 3′-untranslated region of the NKG2D gene and microRNA as well as their functional roles in the regulation of NKG2D expression and cytotoxicity in natural killer cells. Results Transforming growth factor-β1, a major negative regulator of NKG2D expression, post-transcriptionally up-regulated mature microRNA-1245 expression, thus down-regulating NKG2D expression and impairing NKG2D-mediated immune responses in natural killer cells. Conversely, microRNA-1245 down-regulation significantly increased the expression of NKG2D expression in natural killer cells, resulting in more efficient NKG2D-mediated cytotoxicity. Conclusions These results reveal a novel NKG2D regulatory pathway mediated by microRNA-1245, which may represent one of the mechanisms used by transforming growth factor-β1 to attenuate NKG2D expression in natural killer cells. PMID:22491735

Recent insights into the biology of Hodgkin lymphoma: unraveling the mysteries of the Reed-Sternberg cell.

PubMed

Roullet, Michele R; Bagg, Adam

2007-11-01

The microscopic pathology of Hodgkin lymphoma has been recognized for well over a century; however, only in the past 15 years has the enigmatic nature of this peculiar neoplasm been somewhat unraveled. This has been accomplished via a combination of the acquisition, via microdissection, of the prototypically rare malignant cells and their subsequent analysis via a variety of modalities, including genomic studies and expression profiling. This has facilitated the elucidation of the surreptitiously concealed B-cell origin of the cells, their complex but vital relationships with the surrounding micro- and macroenvironment, as well as multiple pathways involved in the pathobiology of this lymphoma. Understanding the intricacies of these intra- and extracellular pathways should allow for the development of less-toxic targeted therapies.

Mixing enhancement by biologically inspired convection in a micro-chamber using alternating current galvanotactic control of the Tetrahymena pyriformis

NASA Astrophysics Data System (ADS)

Kim, Jihoon; Jang, Yonghee; Byun, Doyoung; Hyung Kim, Dal; Jun Kim, Min

2013-09-01

Recently, there has been increasing interest in the swimming behavior of microorganisms and biologically inspired micro-robots. In this study, we investigated biologically induced convection flow with living microorganism using galvanotaxis. We fabricated and evaluated our micro-mixer with motile cells. For the cell based active micro-mixers, two miscible fluids were used to measure the mixing index. Under alternating current (AC) electric fields with varying frequency, a group of motile Tetrahymena pyriformis cells generated reciprocal motion with circulating flows around their pathline, enhancing the mixing ratio.

A micro alkaline direct ethanol fuel cell with platinum-free catalysts

NASA Astrophysics Data System (ADS)

Verjulio, R. W.; Alcaide, F.; Álvarez, G.; Sabaté, N.; Torres-Herrero, N.; Esquivel, J. P.; Santander, J.

2013-11-01

This paper presents the fabrication and characterization of a micro alkaline direct ethanol fuel cell. The device has been conceived as a feasibility demonstrator, using microtechnologies for the fabrication of the current collectors and traditional techniques for the membrane electrode assembly production. The fuel cell works in passive mode, as expected for the simplicity required for micro power systems. Non-noble catalysts have been used in order to implement the main advantage of alkaline systems, showing the feasibility of such a device as a potential very-low-cost power device at mini- and micro scales.

Combined time-lapse cinematography and immuno-electron microscopy.

PubMed

Balfour, B M; Goscicka, T; MacKenzie, J L; Gautam, A; Tate, M; Clark, J

1990-04-01

A method was developed to record interactions between mobile non-adherent immunocytes by time-lapse cinematography and then to study the same cells by immuno-electron microscopy, using monoclonal antibodies against surface components. For this purpose a modified stage was designed to fit an inverted microscope. The attachment included a device to cool the culture chamber with N2 gas, a micro-injector for monoclonal antibody and immuno-gold treatment, and two pairs of washing needles to change the medium without disturbance. The technique was first employed to study the formation of aggregates around the antigen-presenting cells in cultures containing cells from hyper-immunized animals. Recently peripheral blood cells from normal subjects and patients with immune deficiency syndromes were stimulated with pokeweed mitogen, cluster formation was recorded, and the cells were processed for immuno-electron microscopy.

Inhibition of proteinase 3 (PR3) by suramin and fetal calf serum (FCS): effect of PR3 and suramin on Chinese hamster ovary cells (CHO-cells).

PubMed

Karam, Gholamreza Asadi; Rasaee, Mohammad Javad; Mahmoodi, Mehdi; Khaksari, Mohammad

2005-07-01

Proteinase 3 (PR3) is a lysosomal protease that is stored in azurophilic granules neutrophilic granulocytes and monocytes. A number of inhibitors for this proteinase are reported. Comprehensive studies on the inhibitory effect of suramin and heat treated fetal calf serum (deltaFCS) on PR3 have not been reported. It has been reported that PR3 is able to destroy the cytoskeletal integral proteins, but we have not find any reports which showed the effect of this protease on Chinese hamster ovary cells (CHO-cells) in culture medium. Suramin has proven to be useful as an antitumor drug, but there was not any report on the effect of suramin on CHO-cells. The effects of various concentrations of deltaFCS (from 0.5% up to 10%) and suramin (from 0.8 microM up to 100 microM) on PR3 and different concentrations of suramin (from 0.8 microM up to 1000 microM) on CHO-cells were investigated. Data analysis were performed by, Kolmogorov-Smirnov test, ANOVA test and Tukey HSD post tests. Results showed that deltaFCS and suramin have an inhibitory effect on PR3 and these effects increased with increasing the concentration significantly (p < 0.01). PR3 with the concentration of 2.2 Unit/ml has no effect on CHO-cells. Although suramin with the concentration of less than 125 microM cell growth retarded for only a few hours, but with the concentration of 125 to 250 microM inhibit the cell growth for a week, and after that cells gain normal growth gradually. Suramin with concentration of more than 500 microM inhibited the cell growth completely. Although suramin reversibly inhibit the PR3 activity but in concentration of less than 250 microM it had no long-term effect on CHO-cells. Therefore it can be used in the investigation of proteases. There were unknown components in deltaFCS, which cause the inhibition of PR3 activity. This finding is very important in PR3 production in culture medium. However CHO-cells are resistant to PR3 and suramin in low concentration.

Inihibition of Glycolysis by Using a Micro/Nano-Lipid Bromopyruvic Chitosan Carrier as a Promising Tool to Improve Treatment of Hepatocellular Carcinoma

PubMed Central

Dini, Luciana; Citti, Cinzia; Cannazza, Giuseppe

2018-01-01

Glucose consumption in many types of cancer cells, in particular hepatocellular carcinoma (HCC), was followed completely by over-expression of type II hexokinase (HKII). This evidence has been used in modern pharmacotherapy to discover therapeutic target against glycolysis in cancer cells. Bromopyruvate (BrPA) exhibits antagonist property against HKII and can be used to inhibit glycolysis. However, the clinical application of BrPA is mostly combined with inhibition effect for healthy cells particularly erythrocytes. Our strategy is to encapsulate BrPA in a selected vehicle, without any leakage of BrPA out of vehicle in blood stream. This structure has been constructed from chitosan embedded into oleic acid layer and then coated by dual combination of folic acid (FA) and bovine serum albumin (BSA). With FA as specific ligand for cancer folate receptor and BSA that can be an easy binding for hepatocytes, they can raise the potential selection of carrier system. PMID:29320411

Effect of citral and carvacrol on the susceptibility of Listeria monocytogenes and Listeria innocua to antibiotics.

PubMed

Zanini, S F; Silva-Angulo, A B; Rosenthal, A; Rodrigo, D; Martínez, A

2014-05-01

The aim of this study was to evaluate the antibiotic susceptibility of Listeria innocua (L. innocua) and Listeria monocytogenes (L. monocytogenes) cells in the presence of citral and carvacrol at sublethal concentrations in an agar medium. The presence of terpenes in the L. monocytogenes and L. innocua culture medium provided a reduction in the minimal inhibitory concentration (MIC) of all the antibiotics tested. These effects were dependent on the concentration of terpenes present in the culture medium. The combination of citral and carvacrol potentiated antibiotic activity by reducing the MIC values of bacitracin and colistin from 32.0 and 128.0 μg ml⁻¹ to 1.0 and 2.0 μg ml⁻¹, respectively. Thus, both Listeria species became more susceptible to these drugs. In this way, the colistin and bacitracin resistance of L. monocytogenes and L. innocua was reversed in the presence of terpenes. Results obtained in this study show that the phytochemicals citral and carvacrol potentiate antibiotic activity, reducing the MIC values of cultured L. monocytogenes and L. innocua. Phytochemicals citral and carvacrol potentiate antibiotic activity of erythromycin, bacitracin and colistin by reducing the MIC values of cultured Listeria monocytogenes and Listeria innocua. This effect in reducing the MIC values of the antibiotics tested in both micro-organisms was increased when natural antimicrobials were combined. This finding indicated that the combination among terpenes and antibiotic may contribute in reducing the required dosage of antibiotics due to the possible effect of terpenes on permeation barrier of the micro-organism cell membrane. © 2014 The Society for Applied Microbiology.

Clinical Value of Assessing Cytokine Levels for the Differential Diagnosis of Bacterial Meningitis in a Pediatric Population

PubMed Central

Ye, Qing; Shao, Wen-Xia; Shang, Shi-Qiang; Shen, Hong-Qiang; Chen, Xue-Jun; Tang, Yong-Min; Yu, Yong-Lin; Mao, Jian-Hua

2016-01-01

Abstract We performed a prospective observational study to evaluate the utility of measuring inflammatory cytokine levels to discriminate bacterial meningitis from similar common pediatric diseases. Inflammatory cytokine levels and other cerebrospinal fluid (CSF) physicochemical indicators were evaluated in 140 patients who were diagnosed with bacterial meningitis via microbiological culture or PCR assay. The CSF concentrations of interleukin (IL)-6 and IL-10, CSF/blood IL-6 and IL-10 ratios, CSF white blood cell count, and CSF micro total protein were significantly elevated in bacterial meningitis patients compared with healthy children or patients with viral encephalitis, epilepsy, or febrile convulsions (P < 0.001). The area under the curve values for CSF concentrations of IL-6 and IL-10, CSF/blood IL-6 and IL-10 ratios, CSF white blood cell count, and CSF micro total protein to identify bacterial meningitis episodes by receiver-operating characteristic analysis were 0.988, 0.949, 0.995, 0.924, 0.945, and 0.928, respectively. The area under the curve for the combination of CSF IL-6 and CSF/blood IL-6 ratio was larger than that for either parameter alone, and the combination exhibited enhanced specificity and positive predictive value. After effective meningitis treatment, CSF IL-6 levels dropped significantly. These results suggest that CSF IL-6 and CSF/blood IL-6 ratio are good biomarkers in discriminating bacterial meningitis. Evaluating CSF IL-6 and CSF/blood IL-6 ratio in combination can improve diagnostic efficiency. Additionally, CSF IL-6 levels can be used to monitor the effects of bacterial meningitis treatment. PMID:27043692

Clinical Value of Assessing Cytokine Levels for the Differential Diagnosis of Bacterial Meningitis in a Pediatric Population.

PubMed

Ye, Qing; Shao, Wen-Xia; Shang, Shi-Qiang; Shen, Hong-Qiang; Chen, Xue-Jun; Tang, Yong-Min; Yu, Yong-Lin; Mao, Jian-Hua

2016-03-01

We performed a prospective observational study to evaluate the utility of measuring inflammatory cytokine levels to discriminate bacterial meningitis from similar common pediatric diseases. Inflammatory cytokine levels and other cerebrospinal fluid (CSF) physicochemical indicators were evaluated in 140 patients who were diagnosed with bacterial meningitis via microbiological culture or PCR assay. The CSF concentrations of interleukin (IL)-6 and IL-10, CSF/blood IL-6 and IL-10 ratios, CSF white blood cell count, and CSF micro total protein were significantly elevated in bacterial meningitis patients compared with healthy children or patients with viral encephalitis, epilepsy, or febrile convulsions (P < 0.001). The area under the curve values for CSF concentrations of IL-6 and IL-10, CSF/blood IL-6 and IL-10 ratios, CSF white blood cell count, and CSF micro total protein to identify bacterial meningitis episodes by receiver-operating characteristic analysis were 0.988, 0.949, 0.995, 0.924, 0.945, and 0.928, respectively. The area under the curve for the combination of CSF IL-6 and CSF/blood IL-6 ratio was larger than that for either parameter alone, and the combination exhibited enhanced specificity and positive predictive value. After effective meningitis treatment, CSF IL-6 levels dropped significantly. These results suggest that CSF IL-6 and CSF/blood IL-6 ratio are good biomarkers in discriminating bacterial meningitis. Evaluating CSF IL-6 and CSF/blood IL-6 ratio in combination can improve diagnostic efficiency. Additionally, CSF IL-6 levels can be used to monitor the effects of bacterial meningitis treatment.

Etiology of congenital hypothyroidism using thyroglobulin and ultrasound combination.

PubMed

Beltrão, Cristine B; Juliano, Adriana G; Chammas, Maria C; Watanabe, Tomoco; Sapienza, Marcelo T; Marui, Suemi

2010-01-01

Methods currently employed to establish the etiology of congenital hypothyroidism include thyroid ultrasound and scintigraphic exams. Thyroglobulin is a protein almost exclusively secreted by thyroid tissue and indirectly reflects the amount of follicular cells. Even though thyroglobulin is easy to measure, it has been not frequently used because of discordant results to distinguish mainly athyreosis and ectopy (dysgenesis). Knowing the differences in inheritance and prognosis of thyroid dysgenesis and dyshormonogenesis, it is important to define the etiology of CH, combining tools that are easy, fast and available in most medical centers. Our objective was to evaluate and compare color Doppler ultrasound and serum thyroglobulin with radionuclide scan to define the etiology of congenital hypothyroidism. We evaluated 38 children above 3 years-old off-treatment that performed serum thyroglobulin by immunofluorometric assay, color Doppler ultrasound and radionuclide study. On color Doppler ultrasound, 11 patients had athyreosis, 5 ectopic glands, being 1 associated to hemiagenesis. Twenty one had topic thyroid (3 goiters, 10 normal, 8 hypoplastic). Hemiagenesis and cystic lesion were not revealed by radionuclide scan. We observed substantial agreement between color Doppler ultrasound and radionuclide scan (kappa=0.745, p<0.0001). Serum thyroglobulin in athyreosis ranged from <1.0 to 18.7 micro g/L. Patients with ectopic glands showed wider thyroglobulin range (4.5 to 123 micro g/L, median 28.4 micro g/L). Only one patient showed thyroglobulin deficiency. By using color Doppler ultrasound and serum thyroglobulin levels as valuable combined tools, we established the etiology of congenital hypothyroidism limiting excessive and harmful exams in children, like radionuclide scan.

Novel anti-reflection technology for GaAs single-junction solar cells using surface patterning and Au nanoparticles.

PubMed

Kim, Youngjo; Lam, Nguyen Dinh; Kim, Kangho; Kim, Sangin; Rotermund, Fabian; Lim, Hanjo; Lee, Jaejin

2012-07-01

Single-junction GaAs solar cell structures were grown by low-pressure MOCVD on GaAs (100) substrates. Micro-rod arrays with diameters of 2 microm, 5 microm, and 10 microm were fabricated on the surfaces of the GaAs solar cells via photolithography and wet chemical etching. The patterned surfaces were coated with Au nanoparticles using an Au colloidal solution. Characteristics of the GaAs solar cells with and without the micro-rod arrays and Au nanoparticles were investigated. The short-circuit current density of the GaAs solar cell with 2 microm rod arrays and Au nanoparticles increased up to 34.9% compared to that of the reference cell without micro-rod arrays and Au nanoparticles. The conversion efficiency of the GaAs solar cell that was coated with Au nanoparticles on the patterned surface with micro-rod arrays can be improved from 14.1% to 19.9% under 1 sun AM 1.5G illumination. These results show that micro-rod arrays and Au nanoparticle coating can be applied together in surface patterning to achieve a novel cost-effective anti-reflection technology.

Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine resistance by the flavonoid myricetin.

PubMed

van Zanden, Jelmer J; de Mul, Anika; Wortelboer, Heleen M; Usta, Mustafa; van Bladeren, Peter J; Rietjens, Ivonne M C M; Cnubben, Nicole H P

2005-06-01

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5+/-1.7 microM for MRP1 and 24.6+/-1.3 microM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1+/-0.1 microM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1+/-1.9 and 22.2+/-1.4 microM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 microM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6+/-0.5 and 5.8+/-0.5 microM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance.

Comparison of vitamins K1, K2 and K3 effects on growth of rat glioma and human glioblastoma multiforme cells in vitro.

PubMed

Oztopçu, Pinar; Kabadere, Selda; Mercangoz, Ayşe; Uyar, Ruhi

2004-09-01

Glioblastoma multiforme is characterized as highly invasive and rapidly growing astrocytomas, and scientists have sought for efficient treatment against malignant gliomas for a long time. Therefore, we compared the respond of rat glioma (C6) and glioblastoma multiforme cells derived from two patients to vitamins K1, K2 and K3. The cells were exposed to 100, 250, 500, 750 and 1000 microM of vitamins K1 and K2, and 1, 10, 25, 50, 75 and 100 microM of vitamin K3 for 24 hours in an incubator atmosphere of 5% CO2, 37 degrees C and 100% humidity. Cell viability was estimated by MTT assay. Vitamin K1 showed no growth effect on all the glioma cells examined. Vitamin K2 did not cause any change in number of C6, however induced growth inhibition in a dose-dependent manner on glioblastoma multiforme. The IC50 values of vitamin K2 were 960 microM and 970 microM for glioblastoma multiforme, respectively. Vitamin K3 had also growth inhibitory effect in a dose-dependent manner on both C6 and glioblastoma multiforme. The IC50 values were 41 microM, 24 microM and 23 microM for vitamin K3, respectively. We concluded that vitamin K3 is more effective than vitamin K2 for inhibition of cancer cell growth, and might have an alternative value as an anticancer drug against glioblastoma multiforme.

Interference-free Micro/nanoparticle Cell Engineering by Use of High-Throughput Microfluidic Separation.

PubMed

Yeo, David C; Wiraja, Christian; Zhou, Yingying; Tay, Hui Min; Xu, Chenjie; Hou, Han Wei

2015-09-23

Engineering cells with active-ingredient-loaded micro/nanoparticles is becoming increasingly popular for imaging and therapeutic applications. A critical yet inadequately addressed issue during its implementation concerns the significant number of particles that remain unbound following the engineering process, which inadvertently generate signals and impart transformative effects onto neighboring nontarget cells. Here we demonstrate that those unbound micro/nanoparticles remaining in solution can be efficiently separated from the particle-labeled cells by implementing a fast, continuous, and high-throughput Dean flow fractionation (DFF) microfluidic device. As proof-of-concept, we applied the DFF microfluidic device for buffer exchange to sort labeled suspension cells (THP-1) from unbound fluorescent dye and dye-loaded micro/nanoparticles. Compared to conventional centrifugation, the depletion efficiency of free dyes or particles was improved 20-fold and the mislabeling of nontarget bystander cells by free particles was minimized. The microfluidic device was adapted to further accommodate heterogeneous-sized mesenchymal stem cells (MSCs). Complete removal of unbound nanoparticles using DFF led to the usage of engineered MSCs without exerting off-target transformative effects on the functional properties of neighboring endothelial cells. Apart from its effectiveness in removing free particles, this strategy is also efficient and scalable. It could continuously process cell solutions with concentrations up to 10(7) cells·mL(-1) (cell densities commonly encountered during cell therapy) without observable loss of performance. Successful implementation of this technology is expected to pave the way for interference-free clinical application of micro/nanoparticle engineered cells.

Use of multi-functional flexible micro-sensors for in situ measurement of temperature, voltage and fuel flow in a proton exchange membrane fuel cell.

PubMed

Lee, Chi-Yuan; Chan, Pin-Cheng; Lee, Chung-Ju

2010-01-01

Temperature, voltage and fuel flow distribution all contribute considerably to fuel cell performance. Conventional methods cannot accurately determine parameter changes inside a fuel cell. This investigation developed flexible and multi-functional micro sensors on a 40 μm-thick stainless steel foil substrate by using micro-electro-mechanical systems (MEMS) and embedded them in a proton exchange membrane fuel cell (PEMFC) to measure the temperature, voltage and flow. Users can monitor and control in situ the temperature, voltage and fuel flow distribution in the cell. Thereby, both fuel cell performance and lifetime can be increased.

Chemoprevention of Cigarette Smoke–Induced Alterations of MicroRNA Expression in Rat Lungs

PubMed Central

Izzotti, Alberto; Calin, George A.; Steele, Vernon E.; Cartiglia, Cristina; Longobardi, Mariagrazia; Croce, Carlo M.; De Flora, Silvio

2015-01-01

We previously showed that exposure to environmental cigarette smoke (ECS) for 28 days causes extensive downregulation of microRNA expression in the lungs of rats, resulting in the overexpression of multiple genes and proteins. In the present study, we evaluated by microarray the expression of 484 microRNAs in the lungs of either ECS-free or ECS-exposed rats treated with the orally administered chemopreventive agents N-acetylcysteine, oltipraz, indole-3-carbinol, 5,6-benzoflavone, and phenethyl isothiocyanate (as single agents or in combinations). This is the first study of microRNA modulation by chemopreventive agents in nonmalignant tissues. Scatterplot, hierarchical cluster, and principal component analyses of microarray and quantitative PCR data showed that none of the above chemopreventive regimens appreciably affected the baseline microRNA expression, indicating potential safety. On the other hand, all of them attenuated ECS-induced alterations but to a variable extent and with different patterns, indicating potential preventive efficacy. The main ECS-altered functions that were modulated by chemopreventive agents included cell proliferation, apoptosis, differentiation, Ras activation, P53 functions, NF-κB pathway, transforming growth factor–related stress response, and angiogenesis. Some micro-RNAs known to be polymorphic in humans were downregulated by ECS and were protected by chemopreventive agents. This study provides proof-of-concept and validation of technology that we are further refining to screen and prioritize potential agents for continued development and to help elucidate their biological effects and mechanisms. Therefore, microRNA analysis may provide a new tool for predicting at early carcinogenesis stages both the potential safety and efficacy of cancer chemopreventive agents. PMID:20051373

Antiviral activity of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine against human cytomegalovirus in human skin fibroblasts.

PubMed Central

Colacino, J M; Lopez, C

1985-01-01

2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine (FIAC) was shown to be a selective anti-human cytomegalovirus agent in vitro with a 50% antiviral effective dose of 0.6 microM (J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother. 26:505-508, 1983) and a 50% cell growth inhibitory dose of 8 microM. Antiviral activity was more readily reversed with 10-fold excess thymidine, whereby the 50% effective dose was increased to 11.3 microM. FIAC-induced cytotoxicity was more readily reversed with 10-fold excess of deoxycytidine, whereby the 50% inhibitory dose was increased to greater than 100 microM. Thymidine was unable to reverse completely the antiviral activity of FIAC. Although, the extent of phosphorylation of thymidine, deoxycytidine, and deoxyuridine was 6-, 4-, and 4-fold greater, respectively, in human cytomegalovirus-infected cell lysates than in uninfected cell lysates, the extent of phosphorylation of FIAC was only 1.3-fold greater in human cytomegalovirus-infected cell lysates than in uninfected cell lysates. By comparison, the extent of FIAC phosphorylation was 500 times greater in herpes simplex virus type 1-infected cells than in uninfected cell lysates. Methotrexate was 400 times more effective against human cytomegalovirus replication than it was against herpes simplex virus type 1 replication, indicating that thymidylate synthetase may be important for human cytomegalovirus replication. However, 10 microM FIAC did not inhibit thymidylate synthetase activity in uninfected or virus-infected cells as determined by their metabolism of [6-3H]deoxyuridine in the presence or absence of drug. FIAC at 1 microM suppresses and FIAC at 10 microM completely inhibits human cytomegalovirus DNA replication as indicated by Southern blot analysis. This inhibition was reversible. FIAC incorporation into the DNA of human cytomegalovirus strain AD169-infected cells was stimulated relative to that in nondividing, uninfected cells. Images PMID:3010842

Rapid synthesis of VX-745: p38 MAP kinase inhibition in Werner syndrome cells.

PubMed

Bagley, Mark C; Davis, Terence; Dix, Matthew C; Rokicki, Michal J; Kipling, David

2007-09-15

The p38 mitogen-activated protein kinase inhibitor VX-745 is prepared rapidly and efficiently in a four-step sequence using a combination of conductive heating and microwave-mediated steps. Its inhibitory activity was confirmed in hTERT immortalized HCA2 and WS dermal fibroblasts at 0.5-1.0 microM concentration by ELISA and immunoblot assay, and displays excellent kinase selectivity over the related stress-activated kinase JNK.

Origin of Polar Order in Dense Suspensions of Phototactic Micro-Swimmers

PubMed Central

Furlan, Silvano; Comparini, Diego; Ciszak, Marzena; Beccai, Lucia; Mancuso, Stefano; Mazzolai, Barbara

2012-01-01

A main question for the study of collective motion in living organisms is the origin of orientational polar order, i.e., how organisms align and what are the benefits of such collective behaviour. In the case of micro-organisms swimming at a low Reynolds number, steric repulsion and long-range hydrodynamic interactions are not sufficient to explain a homogeneous polar order state in which the direction of motion is aligned. An external symmetry-breaking guiding field such as a mechanism of taxis appears necessary to understand this phonemonon. We have investigated the onset of polar order in the velocity field induced by phototaxis in a suspension of a motile micro-organism, the algae Chlamydomonas reinhardtii, for density values above the limit provided by the hydrodynamic approximation of a force dipole model. We show that polar order originates from a combination of both the external guiding field intensity and the population density. In particular, we show evidence for a linear dependence of a phototactic guiding field on cell density to determine the polar order for dense suspensions and demonstrate the existence of a density threshold for the origin of polar order. This threshold represents the density value below which cells undergoing phototaxis are not able to maintain a homogeneous polar order state and marks the transition to ordered collective motion. Such a transition is driven by a noise dominated phototactic reorientation where the noise is modelled as a normal distribution with a variance that is inversely proportional to the guiding field strength. Finally, we discuss the role of density in dense suspensions of phototactic micro-swimmers. PMID:22723904

Resveratrol glucuronides as the metabolites of resveratrol in humans: characterization, synthesis, and anti-HIV activity.

PubMed

Wang, Lai-Xi; Heredia, Alonso; Song, Haijing; Zhang, Zhaojun; Yu, Biao; Davis, Charles; Redfield, Robert

2004-10-01

Resveratrol is a natural product with diverse biological activities. We have previously reported that resveratrol possesses potent synergistic inhibitory activity against human immunodeficiency virus (HIV)-1 infection in combination with nucleoside analogs (Heredia et al. 2000. J Acquir Immune Defic Syndr 25:246-255). As a part of our program in developing resveratrol as a component for anti-HIV chemotherapy, we describe in this article the characterization, chemical synthesis, and biological effects of the human metabolites of resveratrol. We found that resveratrol was metabolized in humans into two metabolites, which were characterized as resveratrol-3-O- and 4'-O-glucuronides. For further biological studies, we reported two simple, alternative methods for the synthesis of the metabolites. The cytotoxic and antiviral activities of resveratrol and its metabolites were compared in cell culture experiments using human peripheral blood mononuclear cells. Whereas resveratrol was cytotoxic at > or =30 microM, no cytotoxicity was observed for the metabolites at concentrations as high as 300 microM. However, resveratrol showed strong synergistic anti-HIV activity with didanosine at 10 microM, but no synergistic effects were observed for either of the metabolites at up to 300 microM. Nevertheless, the in vitro activity of the metabolites (resveratrol glucuronides) may not necessarily reflect their in vivo function, given the fact that the ubiquitously existing human beta-glucuronidase could convert the metabolites back to resveratrol locally or systematically in vivo. The present studies have implications for future development of resveratrol and/or its derivatives as a chemotherapeutic agent. Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association

Formononetin, an isoflavone, relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways.

PubMed

Wu, Jian-Hong; Li, Qing; Wu, Min-Yi; Guo, De-Jian; Chen, Huan-Le; Chen, Shi-Lin; Seto, Sai-Wang; Au, Alice L S; Poon, Christina C W; Leung, George P H; Lee, Simon M Y; Kwan, Yiu-Wa; Chan, Shun-Wan

2010-07-01

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 microM) and methylene blue (10 microM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS(Ser1177) protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 microM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 microM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 microM, an estrogen receptor (ER alpha/ER beta) antagonist) or mifepristone (10 microM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca(2+)-activated K(+) (BK(Ca)) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K(+) (K(ATP)) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK(Ca) and K(ATP) channels. (c) 2010 Elsevier Inc. All rights reserved.

Combined micro computed tomography and histology study of bone augmentation and distraction osteogenesis

NASA Astrophysics Data System (ADS)

Ilgenstein, Bernd; Deyhle, Hans; Jaquiery, Claude; Kunz, Christoph; Stalder, Anja; Stübinger, Stefan; Jundt, Gernot; Beckmann, Felix; Müller, Bert; Hieber, Simone E.

2012-10-01

Bone augmentation is a vital part of surgical interventions of the oral and maxillofacial area including dental implantology. Prior to implant placement, sufficient bone volume is needed to reduce the risk of peri-implantitis. While augmentation using harvested autologous bone is still considered as gold standard, many surgeons prefer bone substitutes to reduce operation time and to avoid donor site morbidity. To assess the osteogenic efficacy of commercially available augmentation materials we analyzed drill cores extracted before implant insertion. In younger patients, distraction osteogenesis is successfully applied to correct craniofacial deformities through targeted bone formation. To study the influence of mesenchymal stem cells on bone regeneration during distraction osteogenesis, human mesenchymal stem cells were injected into the distraction gap of nude rat mandibles immediately after osteotomy. The distraction was performed over eleven days to reach a distraction gap of 6 mm. Both the rat mandibles and the drill cores were scanned using synchrotron radiation-based micro computed tomography. The three-dimensional data were manually registered and compared with corresponding two-dimensional histological sections to assess bone regeneration and its morphology. The analysis of the rat mandibles indicates that bone formation is enhanced by mesenchymal stem cells injected before distraction. The bone substitutes yielded a wide range of bone volume and degree of resorption. The volume fraction of the newly formed bone was determined to 34.4% in the computed tomography dataset for the augmentation material Geistlich Bio-Oss®. The combination of computed tomography and histology allowed a complementary assessment for both bone augmentation and distraction osteogenesis.

Neurite outgrowth of murine cerebellar granule cells can be enhanced by aniracetam with or without alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA).

PubMed

Fushiki, S; Matsumoto, K; Nagata, A

1995-10-27

To assess the neurotrophic effects of a nootropic drug, aniracetam, we studied neurite extension of mouse cerebellar granule cells in culture with low or with high K+ under different combinations of drugs and then immunohistochemically stained the cells with an antibody against L1, a neural cell adhesion molecule on cerebellar granule cells. Quantitative analyses using parameters of the total neurite length, maximal neurite length and number of branches disclosed that aniracetam, in the presence of high K+ and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), significantly enhanced neurite extension of cultured granule neurons. Aniracetam alone also stimulated neurite extension of cerebellar granule cells at a longer period of culture with low K+ showing a bell-shaped dose response curve with maximal effects at 10 microM. Aniracetam may influence remodeling of the neural network after injury.

Visualization of astaxanthin localization in HT29 human colon adenocarcinoma cells by combined confocal resonance Raman and fluorescence microspectroscopy.

PubMed

Briviba, Karlis; Bornemann, Rainer; Lemmer, Ulrich

2006-11-01

Astaxanthin, a carotenoid found in plants and seafood, exhibits antiproliferative, antioxidant and anticarcinogenic properties. We show that astaxanthin delivered with tetrahydrofuran is effectively taken up by cultured colon adenocarcinoma cells and is localized mostly in the cytoplasm as detected by confocal resonance Raman and broad-band fluorescence microspectroscopy image analysis. Cells incubated with beta-carotene at the same concentration as astaxanthin (10 microM) showed about a 50-fold lower cellular amount of beta-carotene, as detected by HPLC. No detectable Raman signal of beta-carotene was found in cells, but a weak broad-band fluorescence signal of beta-carotene was observed. beta-Carotene, like astaxanthin, was localized mostly in the cytoplasm. The heterogeneity of astaxanthin and beta-carotene cellular distribution in cells of intestinal origin suggests that the possible defense against reactive molecules by carotenoids in these cells may also be heterogeneous.

Polymeric mechanical amplifiers of immune cytokine-mediated apoptosis

NASA Astrophysics Data System (ADS)

Mitchell, Michael J.; Webster, Jamie; Chung, Amanda; Guimarães, Pedro P. G.; Khan, Omar F.; Langer, Robert

2017-03-01

Physical forces affect tumour growth, progression and metastasis. Here, we develop polymeric mechanical amplifiers that exploit in vitro and in vivo physical forces to increase immune cytokine-mediated tumour cell apoptosis. Mechanical amplifiers, consisting of biodegradable polymeric particles tethered to the tumour cell surface via polyethylene glycol linkers, increase the apoptotic effect of an immune cytokine on tumour cells under fluid shear exposure by as much as 50% compared with treatment under static conditions. We show that targeted polymeric particles delivered to tumour cells in vivo amplify the apoptotic effect of a subsequent treatment of immune cytokine, reduce circulating tumour cells in blood and overall tumour cell burden by over 90% and reduce solid tumour growth in combination with the antioxidant resveratrol. The work introduces a potentially new application for a broad range of micro- and nanoparticles to maximize receptor-mediated signalling and function in the presence of physical forces.

Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

PubMed

Song, Linyang; Song, Wei; Schipper, Hyman M

2007-08-01

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage. (c) 2007 Wiley-Liss, Inc.

Evaluation of a moisturising micro-gel spray for prevention of cell dryness in oral mucosal cells: an in vitro study and evaluation in a clinical setting.

PubMed

Ota, Y; Morito, A; Fujisawa, K; Nishida, M; Hata, H; Ueno, T; Yurikusa, T; Murata, T

2012-11-01

A moisturising micro-gel spray for prevention of dryness was compared with commercial products and artificial saliva in vitro and in a clinical setting in patients with cancer. Survival of cultured human gingival epithelial cells was evaluated after treatment with each product for 15 min. A dry test was performed for products giving a 50% survival rate, in which cell survival was measured after drying of cells treated with each product. The survival rates of cells treated with the micro-gel spray and artificial saliva were significantly higher than those of control cells. The micro-gel spray was then evaluated for 1 week in patients with symptoms of dry mouth caused by cancer treatment. There was significant improvement of these symptoms at night and on awakening and of subjective symptoms of decreased salivary volume (P < 0.05). Mean visual analogue scale scores also significantly decreased (P < 0.01). These data suggest that evaluation of moisturising products for dryness prevention can be performed in cultured cells, since products that performed well in vitro also showed good efficacy for symptoms of dry mouth. The micro-gel spray was particularly effective for relieving symptoms of dry mouth in patients with cancer. © 2012 Blackwell Publishing Ltd.

Three-dimensional micro electromechanical system piezoelectric ultrasound transducer

NASA Astrophysics Data System (ADS)

Hajati, Arman; Latev, Dimitre; Gardner, Deane; Hajati, Azadeh; Imai, Darren; Torrey, Marc; Schoeppler, Martin

2012-12-01

Here we present the design and experimental acoustic test data for an ultrasound transducer technology based on a combination of micromachined dome-shaped piezoelectric resonators arranged in a flexible architecture. Our high performance niobium-doped lead zirconate titanate film is implemented in three-dimensional dome-shaped structures, which form the basic resonating cells. Adjustable frequency response is realized by mixing these basic cells and modifying their dimensions by lithography. Improved characteristics such as high sensitivity, adjustable wide-bandwidth frequency response, low transmit voltage compatible with ordinary integrated circuitry, low electrical impedance well matched to coaxial cabling, and intrinsic acoustic impedance match to water are demonstrated.

Combining optical trapping in a microfluidic channel with simultaneous micro-Raman spectroscopy and motion detection

NASA Astrophysics Data System (ADS)

Lawton, Penelope F.; Saunter, Christopher D.; Girkin, John M.

2014-03-01

Since their invention by Ashkin optical tweezers have demonstrated their ability and versatility as a non-invasive tool for micromanipulation. One of the most useful additions to the basic optical tweezers system is micro-Raman spectroscopy, which permits highly sensitive analysis of single cells or particles. We report on the development of a dual laser system combining two spatial light modulators to holographically manipulate multiple traps (at 1064nm) whilst undertaking Raman spectroscopy using a 532nm laser. We can thus simultaneously trap multiple particles and record their Raman spectra, without perturbing the trapping system. The dual beam system is built around micro-fluidic channels where crystallisation of calcium carbonate occurs on polymethylmethacrylate (PMMA) beads. The setup is designed to simulate at a microscopic level the reactions that occur on items in a dishwasher, where permanent filming of calcium carbonate on drinking glasses is a problem. Our system allows us to monitor crystal growth on trapped particles in which the Raman spectrum and changes in movement of the bead are recorded. Due to the expected low level of crystallisation on the bead surfaces this allows us to obtain results quickly and with high sensitivity. The long term goal is to study the development of filming on samples in-situ with the microfl.uidic system acting as a model dishwasher.

Surface modification of polydimethylsiloxane (PDMS) induced proliferation and neural-like cells differentiation of umbilical cord blood-derived mesenchymal stem cells.

PubMed

Kim, Sun-Jung; Lee, Jae Kyoo; Kim, Jin Won; Jung, Ji-Won; Seo, Kwangwon; Park, Sang-Bum; Roh, Kyung-Hwan; Lee, Sae-Rom; Hong, Yun Hwa; Kim, Sang Jeong; Lee, Yong-Soon; Kim, Sung June; Kang, Kyung-Sun

2008-08-01

Stem cell-based therapy has recently emerged for use in novel therapeutics for incurable diseases. For successful recovery from neurologic diseases, the most pivotal factor is differentiation and directed neuronal cell growth. In this study, we fabricated three different widths of a micro-pattern on polydimethylsiloxane (PDMS; 1, 2, and 4 microm). Surface modification of the PDMS was investigated for its capacity to manage proliferation and differentiation of neural-like cells from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). Among the micro-patterned PDMS fabrications, the 1 microm-patterned PDMS significantly increased cell proliferation and most of the cells differentiated into neuronal cells. In addition, the 1 microm-patterned PDMS induced an increase in cytosolic calcium, while the differentiated cells on the flat and 4 microm-patterned PDMS had no response. PDMS with a 1 microm pattern was also aligned to direct orientation within 10 degrees angles. Taken together, micro-patterned PDMS supported UCB-MSC proliferation and induced neural like-cell differentiation. Our data suggest that micro-patterned PDMS might be a guiding method for stem cell therapy that would improve its therapeutic action in neurological diseases.

Effect of clomiphene on Ca(2+) movement in human prostate cancer cells.

PubMed

Jiann, Bang-Ping; Lu, Yih-Chau; Chang, Hong-Tai; Huang, Jong-Khing; Jan, Chung-Ren

2002-05-17

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca(2+) indicator. Clomiphene at concentrations between 10-50 microM increased [Ca(2+)](i) in a concentration-dependent manner. The [Ca(2+)](i) signal was biphasic with an initial rise and a slow decay. Ca(2+) removal inhibited the Ca(2+) signal by 41%. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with clomiphene in Ca(2+)-free medium, confirming that clomiphene induced Ca(2+) entry. In Ca(2+)-free medium, pretreatment with 50 microM brefeldin A (to permeabilize the Golgi complex), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca(2+) pump), and 2 microM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 microM clomiphene-induced store Ca(2+) release. Conversely, pretreatment with 50 microM clomiphene in Ca(2+)-free medium abolished the [Ca(2+)](i) increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 microM clomiphene-induced Ca(2+)release was unaltered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 microM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca(2+)](i) increases in PC3 cells by releasing store Ca(2+) from multiple stores in an phospholipase C-independent manner, and by activating Ca(2+) influx; and clomiphene was of mild cytotoxicity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, Dibash K.; The Graduate Center Departments of Biology and Biochemistry, The City University of New York, New York, NY 10016; Department of Medicine, Weill Cornell Medicine, Cornell University, New York, NY 10065

Prostate cancer (PCa) is frequently diagnosed in men, and dysregulation of microRNAs is characteristic of many cancers. MicroRNA-1207-3p is encoded at the non-protein coding gene locus PVT1 on the 8q24 human chromosomal region, an established PCa susceptibility locus. However, the role of microRNA-1207-3p in PCa is unclear. We discovered that microRNA-1207-3p is significantly underexpressed in PCa cell lines in comparison to normal prostate epithelial cells. Increased expression of microRNA-1207-3p in PCa cells significantly inhibits proliferation, migration, and induces apoptosis via direct molecular targeting of FNDC1, a protein which contains a conserved protein domain of fibronectin (FN1). FNDC1, FN1, and themore » androgen receptor (AR) are significantly overexpressed in PCa cell lines and human PCa, and positively correlate with aggressive PCa. Prostate tumor FN1 expression in patients that experienced PCa-specific death is significantly higher than in patients that remained alive. Furthermore, FNDC1, FN1 and AR are concomitantly overexpressed in metastatic PCa. Consequently, these studies have revealed a novel microRNA-1207-3p/FNDC1/FN1/AR regulatory pathway in PCa. - Graphical abstract: miR-1207-3p/FNDC1/FN1/AR is a novel regulatory pathway in prostate cancer. - Highlights: • Expression of microRNA-1207-3p is significantly lost in prostate cancer (PCa) cells. • MicroRNA-1207-3p regulates proliferation, apoptosis, and migration via direct molecular targeting of the 3′UTR of FNDC1. • MicroRNA-1207-3p regulates proliferation, apoptosis, and migration via direct molecular targeting of the 3′UTR of FNDC1. • FNDC1, FN1, and AR are concurrently overexpressed in metastatic PCa.« less

Effects of fotemustine or dacarbasine on a melanoma cell line pretreated with therapeutic proton irradiation.

PubMed

Ristić-Fira, Aleksandra M; Korićanac, Lela B; Zakula, Jelena J; Valastro, Lucia M; Iannolo, Gioacchin; Privitera, Giuseppe; Cuttone, Giacomo; Petrović, Ivan M

2009-04-09

Considering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed. Cells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak (SOBP). Irradiation doses were 12 or 16 Gy and are those frequently used in proton therapy. Four days after irradiation cells were treated with fotemustine (FM) or dacarbazine (DTIC). Drug concentrations were 100 and 250 microM, values close to those that produce 50% of growth inhibition. Cell viability, proliferation, survival and cell cycle distribution were assessed 7 days after irradiation that corresponds to more than six doubling times of HTB140 cells. In this way incubation periods providing the best single effects of drugs (3 days) and protons (7 days) coincided at the same time. Single proton irradiations have reduced the number of cells to approximately 50%. FM caused stronger cell inactivation due to its high toxicity, while the effectiveness of DTIC, that was important at short term, almost vanished with the incubation of 7 days. Cellular mechanisms triggered by proton irradiation differently influenced the final effects of combined treatments. Combination of protons and FM did not improve cell inactivation level achieved by single treatments. A low efficiency of the single DTIC treatment was overcome when DTIC was introduced following proton irradiation, giving better inhibitory effects with respect to the single treatments. Most of the analysed cells were in G1/S phase, viable, active and able to replicate DNA. The obtained results are the consequence of a high resistance of HTB140 melanoma cells to protons and/or drugs. The inactivation level of the HTB140 human melanoma cells after protons, FM or DTIC treatments was not enhanced by their combined application.

In vitro synchrotron-based radiography of micro-gap formation at the implant-abutment interface of two-piece dental implants.

PubMed

Rack, A; Rack, T; Stiller, M; Riesemeier, H; Zabler, S; Nelson, K

2010-03-01

Micro-gap formation at the implant-abutment interface of two-piece dental implants was investigated in vitro using high-resolution radiography in combination with hard X-ray synchrotron radiation. Images were taken with the specimen under different mechanical loads of up to 100 N. The aim of this investigation was to prove the existence of micro-gaps for implants with conical connections as well as to study the mechanical behavior of the mating zone of conical implants during loading. Synchrotron-based radiography in comparison with classical laboratory radiography yields high spatial resolution in combination with high contrast even when exploiting micro-sized features in highly attenuating objects. The first illustration of a micro-gap which was previously indistinguishable by laboratory methods underlines that the complex micro-mechanical behavior of implants requires further in vitro investigations where synchrotron-based micro-imaging is one of the prerequisites.

Global microRNA profiling of peripheral blood mononuclear cells in patients with Behçet's disease.

PubMed

Erre, Gian Luca; Piga, Matteo; Carru, Ciriaco; Angius, Andrea; Carcangiu, Laura; Piras, Marco; Sotgia, Salvatore; Zinellu, Angelo; Mathieu, Alessandro; Passiu, Giuseppe; Pescatori, Mario

2015-01-01

To explore the post-transcriptional regulation of the peripheral blood mononuclear cells (PBMCs) transcriptome by microRNAs in Behçet's disease (BD). Using TaqMan Low Density Array-based microRNAs expression profiling, the expression of 750 mature human microRNAs in PBMCs from 5 BD patients and 3 healthy controls (HC) was compared. The expression of deregulated microRNAs was then validated by quantitative real time-polymerase chain reaction (qRT-PCR), in 42 BD patients and 8 HC. In the initial screening, 13 microRNAs appeared deregulated in BD vs HC. Among them, the differential expression of miR-720 and miR-139-3p was confirmed by qRT-PCR, (p<0.05 and FDR<5%). Areas under the receiver operating characteristic curve for miR-139-3p, miR-720 and miR-139-3p+miR-720 in the validation cohort were 0.84, 0.87 and 0.92 respectively, indicating good discrimination between BD patients and HC. Post-hoc analysis showed that 9 out of 13 microRNAs from the discovery phase were significantly upregulated in active vs. quiescent BD, suggesting inflammation as a key regulator of microRNAs machinery in BD. In silico analysis revealed that several BD candidate susceptibility genes are predicted target of significantly deregulated microRNAs in active BD. A significant enrichment in microRNAs targeting elements of the Toll-like receptor (TLR) and T-cell receptor signalling pathways was also assumed. miR199-3p and miR720 deserve further confirmation as biomarkers of BD in larger studies. PBMCs from active BD displayed a unique signature of microRNAs which may be implicated in regulation of innate immunity activation and T-cell function.

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs.

PubMed

Silva, Karina R; Rezende, Rodrigo A; Pereira, Frederico D A S; Gruber, Peter; Stuart, Mellannie P; Ovsianikov, Aleksandr; Brakke, Ken; Kasyanov, Vladimir; da Silva, Jorge V L; Granjeiro, José M; Baptista, Leandra S; Mironov, Vladimir

2016-01-01

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young's modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p › 0,999) and gene fold expression for SOX-9 and RUNX2 (p › 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.

Perfusion directed 3D mineral formation within cell-laden hydrogels.

PubMed

Sawyer, Stephen William; Shridhar, Shivkumar Vishnempet; Zhang, Kairui; Albrecht, Lucas; Filip, Alex; Horton, Jason; Soman, Pranav

2018-06-08

Despite the promise of stem cell engineering and the new advances in bioprinting technologies, one of the major challenges in the manufacturing of large scale bone tissue scaffolds is the inability to perfuse nutrients throughout thick constructs. Here, we report a scalable method to create thick, perfusable bone constructs using a combination of cell-laden hydrogels and a 3D printed sacrificial polymer. Osteoblast-like Saos-2 cells were encapsulated within a gelatin methacrylate (GelMA) hydrogel and 3D printed polyvinyl alcohol (PVA) pipes were used to create perfusable channels. A custom-built bioreactor was used to perfuse osteogenic media directly through the channels in order to induce mineral deposition which was subsequently quantified via microCT. Histological staining was used to verify mineral deposition around the perfused channels, while COMSOL modeling was used to simulate oxygen diffusion between adjacent channels. This information was used to design a scaled-up construct containing a 3D array of perfusable channels within cell-laden GelMA. Progressive matrix mineralization was observed by cells surrounding perfused channels as opposed to random mineral deposition in static constructs. MicroCT confirmed that there was a direct relationship between channel mineralization within perfused constructs and time within the bioreactor. Furthermore, the scalable method presented in this work serves as a model on how large-scale bone tissue replacement constructs could be made using commonly available 3D printers, sacrificial materials, and hydrogels. © 2018 IOP Publishing Ltd.

Induction of cytogenetic damage in human lymphocytes in vitro and of antineoplastic effects in Ehrlich ascites tumor cells in vivo treated by methotrexate, hyperthermia and/or caffeine.

PubMed

Maskaleris, T; Lialiaris, T; Triantaphyllidis, C

1998-12-03

The synergistic effect of methotrexate (at concentrations between 3. 1 and 100 nM) and its combinations with caffeine (618 microM) and/or hyperthermia (42 degreesC for 2 h) on the frequency of sister chromatid exchanges (SCEs), the proliferating rate index and the mitotic index in cultured human lymphocytes, was examined. Also, the in vivo antineoplastic effects of methotrexate (at a concentration of 0.45 microg/g body weight) and its combination with caffeine (120 microg/g body weight), both on the survival time and the increase of the weight of tumor of BALB/c mice inoculated with Ehrlich ascites tumor cells was examined in the present study. The results indicated that: (a) the triple combination of methotrexate, caffeine and hyperthermia synergistically increased the levels of SCEs and exerted cytostatic and cytotoxic action and (b) the combination of methotrexate and caffeine significantly increased the survival span of the mice inoculated with Ehrlich ascites tumor cells and reduced the increase of the weight of their tumors at rates higher than in the case of methotrexate by itself. It is suggested that the above triple combination (methotrexate plus caffeine plus hyperthermia) could achieve increased effectiveness of methotrexate, better therapy results, and could be successfully applied in the treatment of various types of cancer. Copyright 1998 Elsevier Science B.V.

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

PubMed

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.

Advanced Sensor and Packaging Technologies for Intelligent Adaptive Engine Controls (Preprint)

DTIC Science & Technology

2013-05-01

combination of micro-electromechanical systems (MEMS) sensor technology, novel ceramic materials, high - temperature electronics, and advanced harsh...with simultaneous pressure measurements up to 1,000 psi. The combination of a high - temperature , high -pressure-ratio compressor system, and adaptive...combination of micro-electromechanical systems (MEMS) sensor technology, novel ceramic materials, high temperature electronics, and advanced harsh

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

PubMed

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun; Chang, Po-Ling

2017-09-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target ( HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

Microfluidics microFACS for Life Detection

NASA Technical Reports Server (NTRS)

Platt, Donald W.; Hoover, Richard B.

2010-01-01

A prototype micro-scale Fluorescent Activated Cell Sorter (microFACS) for life detection has been built and is undergoing testing. A functional miniature microfluidics instrument with the ability to remotely distinguish live or dead bacterial cells from abiotic particulates in ice or permafrost of icy bodies of the solar system would be of fundamental value to NASA. The use of molecular probes to obtain the bio-signature of living or dead cells could answer the most fundamental question of Astrobiology: Does life exist beyond Earth? The live-dead fluorescent stains to be used in the microFACS instrument function only with biological cell walls. The detection of the cell membranes of living or dead bacteria (unlike PAH's and many other Biomarkers) would provide convincing evidence of present or past life. This miniature device rapidly examine large numbers of particulates from a polar ice or permafrost sample and distinguish living from dead bacteria cells and biological cells from mineral grains and abiotic particulates and sort the cells and particulates based on a staining system. Any sample found to exhibit fluorescence consistent with living cells could then be used in conjunction with a chiral labeled release experiment or video microscopy system to seek addition evidence for cellular metabolism or motility. Results of preliminary testing and calibration of the microFACS prototype instrument system with pure cultures and enrichment assemblages of microbial extremophiles will be reported.

Combination of doxorubicin and low-intensity ultrasound causes a synergistic enhancement in cell killing and an additive enhancement in apoptosis induction in human lymphoma U937 cells.

PubMed

Yoshida, Toru; Kondo, Takashi; Ogawa, Ryohei; Feril, Loreto B; Zhao, Qing-Li; Watanabe, Akihiko; Tsukada, Kazuhiro

2008-04-01

Potential clinical use of ultrasound (US) in enhancing the effects of anticancer drugs in the treatment of cancers has been highlighted in previous reports. Increased uptake of drugs by the cancer cells due to US has been suggested as a mechanism. However, the precise mechanism of the enhancement has not yet been elucidated. Here, the combined effects of low-intensity pulsed US and doxorubicin (DOX) on cell killing and apoptosis induction of U937 cells, and mechanisms involved were investigated. Human myelomonocytic lymphoma U937 cells were used for the experiments. Experiments were conducted in 4 groups: (1) non-treated, (2) DOX treated (DOX), (3) US treated (US), and (4) combined (DOX + US). In DOX +US, cells were exposed to 5 microM DOX for 30 min and sonicated by 1 MHz pulsed US (PRF 100 Hz, DF 10%) at intensities of 0.2-0.5 W/cm(2) for 60 s. The cells were washed and incubated for 6 h. The viability was evaluated by Trypan blue dye exclusion test and apoptosis and incorporation of DOX was assessed by flow cytometry. Involvement of sonoporation in molecular incorporation was evaluated using FITC-dextran, hydroxyl radical formation was measured by electron paramagnetic resonance-spin trapping, membrane alteration including lipid peroxidation and membrane fluidity by DOX was evaluated using cis-parinaric acid and perylene fluorescence polarization method, respectively. Synergistic enhancement in cell killing and additive enhancement in induction of apoptosis were observed at and above 0.3 W/cm(2). No enhancement was observed at 0.2 W/cm(2) in cell killing and induction of apoptosis. Hydroxyl radicals formation was detected at and above 0.3 W/cm(2). The radicals were produced more in the DOX + US than US alone. Incorporation of DOX was increased 13% in DOX + US (vs. DOX) at 0.5 W/cm(2). Involvement of sonoporation for increase of drug uptake was suggested by experiment using FITC-labeled dextran. We made the hypothesis that DOX treatment made the cells weaken against the mechanical effect of the US. Although treatment of DOX at 5 microM for 30 min did not affect lipid peroxidation and fluidity of cell membrane significantly, higher concentration and longer treatment of DOX induced the significant alteration of cell membrane. Mechanisms of enhancements could be (1) increase in incorporation of the DOX by US involved with sonoporation, (2) enhancement of the cavitation by DOX. Cavitation is required for the enhancement of the effect of DOX. Although the precise involvement of the membrane modifications by DOX in the enhancement remains to be elucidated, they could be involved in the latent effects.

Neutropenia associated with vincristine and L-asparaginase induction chemotherapy for canine lymphoma.

PubMed

Northrup, Nicole C; Rassnick, Kenneth M; Snyder, Laura A; Stone, Michael S; Kristal, Orna; Cotter, Susan M; Moore, Antony S

2002-01-01

Vincristine (VCR) and L-asparaginase (L-ASP) are commonly used to treat canine lymphoma. As single agents, these drugs are not myelosuppressive. However, in combination, VCR and L-ASP cause severe neutropenia in some dogs. It has been recommended that L-ASP be administered 12-24 hours after VCR to minimize toxicity. The purpose of this retrospective study was to determine the prevalence of neutropenia after VCR/L-ASP induction therapy for canine lymphoma and to evaluate risk factors for myelosuppression, especially the interval between VCR and L-ASP administration. Medical records of 147 dogs were reviewed. L-ASP was given 0 (n = 50), 6 (n = 23), 18 (n = 20), or 24 (n = 54) hours after VCR. Forty percent of the dogs were neutropenic 7 days after VCR/L-ASP, and 18% had neutrophil counts of <1,000 cells/microL. The median neutrophil count was 3,712 cells/microL (range 0-30,968 cells/microL). No correlation was found between administration interval and day 7 neutrophil count (P = .84) or development of gastrointestinal signs, including vomiting (P = .80), diarrhea (P = .52), and decreased appetite (P = .30). No significant predictors of neutropenia were identified. Higher clinical stage and substage b were associated with decreased appetite after treatment (P = .04 and .01, respectively). Sixteen percent of the dogs were hospitalized. This study demonstrates that VCR/L-ASP induction for canine lymphoma may result in neutropenia but that separation of VCR and L-ASP administration may not be necessary to avoid toxicity.

Effects of chromium(III) picolinate on cortisol and DHEAs secretion in H295R human adrenocortical cells.

PubMed

Kim, Beob G; Adams, Julye M; Jackson, Brian A; Lindemann, Merlin D

2010-02-01

Dietary chromium(III) picolinate (CrPic) effects on circulating steroid hormones have been reported in various experimental animals. However, direct effects of CrPic on adrenocortical steroidogenesis are uncertain. Therefore, the objective was to determine the effects of CrPic on cortisol and dehydroepiandrosterone sulfate (DHEAs) secretion from H295R cells. In experiment 1, a 24-h exposure to CrPic (0 to 200 microM) had both linear (p < 0.001) and quadratic (p < 0.001) effects on cortisol secretion from forskolin-stimulated cells with the highest cortisol secretion at 0.1 microM of CrPic and the lowest at 200 microM of CrPic. In experiment 2, a 48-h exposure to CrPic (200 microM) decreased cortisol (p < 0.07) release from forskolin-stimulated cells during a 24-h collection period. In experiment 3, a 48-h exposure to CrPic (100 microM) decreased cortisol (p < 0.05) and DHEAs (p < 0.01) from forskolin-stimulated cells during a 24-h sampling period. In experiment 4, a 24-h exposure to forskolin followed by a 24-h exposure to both forskolin and CrPic (100 and 200 microM) decreased both cortisol and DHEAs secretion (p < 0.01). This study suggests that at high concentrations, CrPic inhibits aspects of steroidogenesis in agonist-stimulated adrenocortical cells.

Complementary approaches to diagnosing marine diseases: a union of the modern and the classic

PubMed Central

Burge, Colleen A.; Friedman, Carolyn S.; Getchell, Rodman; House, Marcia; Mydlarz, Laura D.; Prager, Katherine C.; Renault, Tristan; Kiryu, Ikunari; Vega-Thurber, Rebecca

2016-01-01

Linking marine epizootics to a specific aetiology is notoriously difficult. Recent diagnostic successes show that marine disease diagnosis requires both modern, cutting-edge technology (e.g. metagenomics, quantitative real-time PCR) and more classic methods (e.g. transect surveys, histopathology and cell culture). Here, we discuss how this combination of traditional and modern approaches is necessary for rapid and accurate identification of marine diseases, and emphasize how sole reliance on any one technology or technique may lead disease investigations astray. We present diagnostic approaches at different scales, from the macro (environment, community, population and organismal scales) to the micro (tissue, organ, cell and genomic scales). We use disease case studies from a broad range of taxa to illustrate diagnostic successes from combining traditional and modern diagnostic methods. Finally, we recognize the need for increased capacity of centralized databases, networks, data repositories and contingency plans for diagnosis and management of marine disease. PMID:26880839

Complementary approaches to diagnosing marine diseases: a union of the modern and the classic

USGS Publications Warehouse

Burge, Colleen A.; Friedman, Carolyn S.; Getchell, Rodman G.; House, Marcia; Lafferty, Kevin D.; Mydlarz, Laura D.; Prager, Katherine C.; Sutherland, Kathryn P.; Renault, Tristan; Kiryu, Ikunari; Vega-Thurber, Rebecca

2016-01-01

Linking marine epizootics to a specific aetiology is notoriously difficult. Recent diagnostic successes show that marine disease diagnosis requires both modern, cutting-edge technology (e.g. metagenomics, quantitative real-time PCR) and more classic methods (e.g. transect surveys, histopathology and cell culture). Here, we discuss how this combination of traditional and modern approaches is necessary for rapid and accurate identification of marine diseases, and emphasize how sole reliance on any one technology or technique may lead disease investigations astray. We present diagnostic approaches at different scales, from the macro (environment, community, population and organismal scales) to the micro (tissue, organ, cell and genomic scales). We use disease case studies from a broad range of taxa to illustrate diagnostic successes from combining traditional and modern diagnostic methods. Finally, we recognize the need for increased capacity of centralized databases, networks, data repositories and contingency plans for diagnosis and management of marine disease.

Use of Multi-Functional Flexible Micro-Sensors for in situ Measurement of Temperature, Voltage and Fuel Flow in a Proton Exchange Membrane Fuel Cell

PubMed Central

Lee, Chi-Yuan; Chan, Pin-Cheng; Lee, Chung-Ju

2010-01-01

Temperature, voltage and fuel flow distribution all contribute considerably to fuel cell performance. Conventional methods cannot accurately determine parameter changes inside a fuel cell. This investigation developed flexible and multi-functional micro sensors on a 40 μm-thick stainless steel foil substrate by using micro-electro-mechanical systems (MEMS) and embedded them in a proton exchange membrane fuel cell (PEMFC) to measure the temperature, voltage and flow. Users can monitor and control in situ the temperature, voltage and fuel flow distribution in the cell. Thereby, both fuel cell performance and lifetime can be increased. PMID:22163545

Mammalian cells loaded with platinum-containing molecules are sensitized to fast atomic ions.

PubMed

Usami, N; Furusawa, Y; Kobayashi, K; Lacombe, S; Reynaud-Angelin, A; Sage, E; Wu, Ting-Di; Croisy, A; Guerquin-Kern, J-L; Le Sech, C

2008-07-01

This work investigates whether a synergy in cell death induction exists in combining atomic ions irradiation and addition of platinum salts. Such a synergy could be of interest in view of new cancer therapy protocol based on atomic ions--hadrontherapy--with the addition of radiosensitizing agents containing high-Z atoms. The experiment consists in irradiating by fast ions cultured cells previously exposed to dichloroterpyridine Platinum (PtTC) and analyzing cell survival by a colony-forming assay. Chinese Hamster Ovary (CHO) cells were incubated for six hours in medium containing 350 microM PtTC, and then irradiated by fast ions C(6+) and He(2+), with Linear Energy Transfer (LET) within range 2-70 keV/microm. In some experiments, dimethyl sulfoxide (DMSO) was added to investigate the role of free radicals. The intracellular localization of platinum was determined by Nano Secondary Ion Mass Spectroscopy (Nano-SIMS). For all LET examined, cell death rate is largely enhanced when irradiating in presence of PtTC. At fixed irradiation dose, cell death rate increases with increasing LET, while the platinum relative effect is larger at low LET. This finding suggests that hadrontherapy or protontherapy therapeutic index could be improved by combining irradiation procedure with concomitant chemotherapy protocols using platinum salts.

Fabrication of a multi-layer three-dimensional scaffold with controlled porous micro-architecture for application in small intestine tissue engineering.

PubMed

Knight, Toyin; Basu, Joydeep; Rivera, Elias A; Spencer, Thomas; Jain, Deepak; Payne, Richard

2013-01-01

Various methods can be employed to fabricate scaffolds with characteristics that promote cell-to-material interaction. This report examines the use of a novel technique combining compression molding with particulate leaching to create a unique multi-layered scaffold with differential porosities and pore sizes that provides a high level of control to influence cell behavior. These cell behavioral responses were primarily characterized by bridging and penetration of two cell types (epithelial and smooth muscle cells) on the scaffold in vitro. Larger pore sizes corresponded to an increase in pore penetration, and a decrease in pore bridging. In addition, smaller cells (epithelial) penetrated further into the scaffold than larger cells (smooth muscle cells). In vivo evaluation of a multi-layered scaffold was well tolerated for 75 d in a rodent model. This data shows the ability of the components of multi-layered scaffolds to influence cell behavior, and demonstrates the potential for these scaffolds to promote desired tissue outcomes in vivo.

MEMS-based thin-film fuel cells

DOEpatents

Jankowksi, Alan F.; Morse, Jeffrey D.

2003-10-28

A micro-electro-mechanical systems (MEMS) based thin-film fuel cells for electrical power applications. The MEMS-based fuel cell may be of a solid oxide type (SOFC), a solid polymer type (SPFC), or a proton exchange membrane type (PEMFC), and each fuel cell basically consists of an anode and a cathode separated by an electrolyte layer. Additionally catalyst layers can also separate the electrodes (cathode and anode) from the electrolyte. Gas manifolds are utilized to transport the fuel and oxidant to each cell and provide a path for exhaust gases. The electrical current generated from each cell is drawn away with an interconnect and support structure integrated with the gas manifold. The fuel cells utilize integrated resistive heaters for efficient heating of the materials. By combining MEMS technology with thin-film deposition technology, thin-film fuel cells having microflow channels and full-integrated circuitry can be produced that will lower the operating temperature an will yield an order of magnitude greater power density than the currently known fuel cells.

MicroRNA let-7d regulates the TLX/microRNA-9 cascade to control neural cell fate and neurogenesis

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Ye, Peng; Li, Shengxiu; Shi, Yanhong

2013-01-01

MicroRNAs have important functions in the nervous system through post-transcriptional regulation of neurogenesis genes. Here we show that microRNA let-7d, which has been implicated in cocaine addiction and other neurological disorders, targets the neural stem cell regulator TLX. Overexpression of let-7d in vivo reduced neural stem cell proliferation and promoted premature neuronal differentiation and migration, a phenotype similar to those induced by TLX knockdown or overexpression of its negatively-regulated target, microRNA-9. We found a let-7d binding sequence in the tlx 3′ UTR and demonstrated that let-7d reduced TLX expression levels in neural stem cells, which in turn, up-regulated miR-9 expression. Moreover, co-expression of let-7d and TLX lacking its 3′ UTR in vivo restored neural stem cell proliferation and reversed the premature neuronal differentiation and migration. Therefore, manipulating let-7d and its downstream targets could be a novel strategy to unravel neurogenic signaling pathways and identify potential interventions for relevant neurological disorders. PMID:23435502

MicroRNA let-7d regulates the TLX/microRNA-9 cascade to control neural cell fate and neurogenesis.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Ye, Peng; Li, Shengxiu; Shi, Yanhong

2013-01-01

MicroRNAs have important functions in the nervous system through post-transcriptional regulation of neurogenesis genes. Here we show that microRNA let-7d, which has been implicated in cocaine addiction and other neurological disorders, targets the neural stem cell regulator TLX. Overexpression of let-7d in vivo reduced neural stem cell proliferation and promoted premature neuronal differentiation and migration, a phenotype similar to those induced by TLX knockdown or overexpression of its negatively-regulated target, microRNA-9. We found a let-7d binding sequence in the tlx 3' UTR and demonstrated that let-7d reduced TLX expression levels in neural stem cells, which in turn, up-regulated miR-9 expression. Moreover, co-expression of let-7d and TLX lacking its 3' UTR in vivo restored neural stem cell proliferation and reversed the premature neuronal differentiation and migration. Therefore, manipulating let-7d and its downstream targets could be a novel strategy to unravel neurogenic signaling pathways and identify potential interventions for relevant neurological disorders.

Cell death induced by flavonoid glycosides with and without copper.

PubMed

Hsu, Hsue-Yin; Tsang, Shih-Fang; Lin, Kai-Wei; Yang, Shyh-Chyun; Lin, Chun-Nan

2008-07-01

The ability of flavonoid glycosides isolated from several plants to induce DNA breakage was examined using supercoiled plasmid pBR322 DNA by agarose gel electrophoresis in the presence of Cu(II). Among all the compounds, 1, 4, and 6 could cause significant breakages of supercoiled plasmid pBR322 DNA in the presence of Cu(II). Cu(I) was not shown to be an essential intermediate in the process of pBR322 DNA breakage by using the Cu(I)-specific sequestering reagent neocuproine. A decreased cell viability was enhanced in gastric carcinoma SCM-1 cells treating with lower concentrations of 1 and 6 when cotreated with increased concentrations of Cu(II), respectively. Treatments of SCM-1 cells with 500 microM of 1 in the presence of 300 or 500 microM of Cu(II) inhibited the Cu(II)-induced apoptosis. Compound 1 (500 microM) could prevent cell death by inhibiting the 500 microM Cu(II)-induced apoptosis and necrosis, but did not have any effect on the mitochondrial membrane potential changed by 500 microM Cu(II). Both compounds 1 and 6 could inhibit the DNA breakages caused by O2- while 1 also revealed inhibitory effect on xanthine oxidase with an IC50 value of 22.7+/-6.9 microM. These results indicated that compound 1 with a higher concentration may probably mediate through the suppression of xanthine oxidase activity and reduce reactive oxygen species (ROS) induced by high concentration of Cu(II) (500 microM) and prevent the following cell death.

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.

PubMed

Liu, Can; Kelnar, Kevin; Liu, Bigang; Chen, Xin; Calhoun-Davis, Tammy; Li, Hangwen; Patrawala, Lubna; Yan, Hong; Jeter, Collene; Honorio, Sofia; Wiggins, Jason F; Bader, Andreas G; Fagin, Randy; Brown, David; Tang, Dean G

2011-02-01

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.

Hetero-cellular prototyping by synchronized multi-material bioprinting for rotary cell culture system.

PubMed

Snyder, Jessica; Son, Ae Rin; Hamid, Qudus; Wu, Honglu; Sun, Wei

2016-01-13

Bottom-up tissue engineering requires methodological progress of biofabrication to capture key design facets of anatomical arrangements across micro, meso and macro-scales. The diffusive mass transfer properties necessary to elicit stability and functionality require hetero-typic contact, cell-to-cell signaling and uniform nutrient diffusion. Bioprinting techniques successfully build mathematically defined porous architecture to diminish resistance to mass transfer. Current limitations of bioprinted cell assemblies include poor micro-scale formability of cell-laden soft gels and asymmetrical macro-scale diffusion through 3D volumes. The objective of this work is to engineer a synchronized multi-material bioprinter (SMMB) system which improves the resolution and expands the capability of existing bioprinting systems by packaging multiple cell types in heterotypic arrays prior to deposition. This unit cell approach to arranging multiple cell-laden solutions is integrated with a motion system to print heterogeneous filaments as tissue engineered scaffolds and nanoliter droplets. The set of SMMB process parameters control the geometric arrangement of the combined flow's internal features and constituent material's volume fractions. SMMB printed hepatocyte-endothelial laden 200 nl droplets are cultured in a rotary cell culture system (RCCS) to study the effect of microgravity on an in vitro model of the human hepatic lobule. RCCS conditioning for 48 h increased hepatocyte cytoplasm diameter 2 μm, increased metabolic rate, and decreased drug half-life. SMMB hetero-cellular models present a 10-fold increase in metabolic rate, compared to SMMB mono-culture models. Improved bioprinting resolution due to process control of cell-laden matrix packaging as well as nanoliter droplet printing capability identify SMMB as a viable technique to improve in vitro model efficacy.

Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures.

PubMed

Heinrich, A; Balszuweit, F; Thiermann, H; Kehe, K

2009-12-15

Sulfur mustard (SM; bis(2-chloroethyl)sulphide; HD) is a blister inducing agent causing DNA damage and subsequently, cell death, mostly by apoptosis in basal keratinocytes. Despite intensive investigations on the cellular mechanism, there are, as of now, no causal therapeutics to prevent or antagonize SM-related damage to cells and tissues. In order to develop treatment strategies against vesication, it is important to distinguish apoptosis from necrosis in SM treated human keratinocytes. DNA fragmentation is a hallmark of apoptosis and regulated by a cascade of enzymes (endonucleases, DNase I, NUC 18), which finally cut the chromatin into specific formations of 180-200 base pairs, the nucleosomes. A feasible way to monitor apoptosis is the detection of nucleosomes by means of the Cell Death Detection ELISA(plus) (CDDE). In contrast, during necrosis DNA fragmentation is at random and delivers larger fragments, which therefore are significantly less in number and predominantly occur in cell culture supernatant. To monitor necrosis, we measured the release of intracellular adenylate kinase (AK) into cell culture supernatant by means of the ToxiLight Bioluminescence Assay (TL). With combination of the Cell Death Detection ELISA(plus) and the ToxiLight Bioluminescence Assay, we acquired more comprehensive information on cell survival and mechanisms of cell death, following an SM exposure. To validate the assay we tested common apoptosis- and necrosis-inducing agents like SM 300 microM for 30 min, Lewisite (L) 60 microM for 5 min and Triton X-100 0.1%. The results show that it is possible to differentiate between the two modes of cell death and to quantify their extent. This assay is highly effective in quantifying apoptosis and necrosis caused by cytotoxic agents and in estimating protective effects of potential active pharmaceutical ingredients.

Nicergoline stimulates protein kinase C mediated alpha-secretase processing of the amyloid precursor protein in cultured human neuroblastoma SH-SY5Y cells.

PubMed

Cedazo-Minguez, A; Bonecchi, L; Winblad, B; Post, C; Wong, E H; Cowburn, R F; Benatti, L

1999-10-01

We investigated the ability of the antidementia agents, nicergoline, aniracetam and hydergine to stimulate PKC mediated alpha-secretase amyloid precursor protein (APP) processing in cultured human neuroblastoma SH-SY5Y cells. Western immunoblotting of cell conditioned media using the Mabs 22C11 and 6E10 revealed the presence of 2 bands with molecular mass of 90 and 120 kDa, corresponding to possible alternatively glycosylated forms of secreted APP (APPs). Short-term (30 min and 2 h) treatment of cells with nicergoline gave an increased intensity of both bands, compared to non-treated cells. Maximal nicergoline effects, of the order of 150-200% over basal APPs release, were seen at concentrations between 1 and 10 microM. Under the same condition, 1 microM PdBu, used as a positive control, gave 500-1000% increases of basal APPs release. In contrast, aniracetam and hydergine, did not show any effect on APPs secretion. 2 h treatment with nicergoline had no effect on cellular full-length APP levels, as determined by immunoblotting of cell extracts with 22C11 and CT15 antibodies. Immunoblotting with PKC isoform specific antibodies of soluble and membrane fractions prepared from 2 h treated cells, showed that nicergoline (50 microM) and PdBu (1 microM) both induced translocation of PKC alpha, gamma and epsilon, but not PKC beta. The involvement of PKC in mediating nicergoline stimulated APPs release was also studied using specific inhibitors. 1 microM calphostin C, a broad range PKC inhibitor, significantly reduced both PdBu (1 microM) and nicergoline (10 microM) induced APPs release. In contrast, Go6976 (1 microM), a selective PKC alpha and beta1 inhibitor, as well as the cAMP-dependent protein kinase inhibitor, H89 (1 microM) were without effect. These results indicate that nicergoline can modulate alpha-secretase APP processing by a PKC dependent mechanism that is likely to involve the gamma and epsilon isoforms of this enzyme.

Integrated Micro-scale Power Conversion

DTIC Science & Technology

2012-08-01

Micro Power Converters (μPC) Loads: Sources: μ-Power Converter (μPC) Thin-film battery Solar Cell Micro- fuel Cell Vibration Harvester...passive size • Hybrid integration with MEMS passives, particularly inductors Hybrid integration ARL focus Bubble Size = Volume [mm3] Industry Focus...Power converters survey Compiled by Bedair, Bashirullah Switched inductor (SI) Switched capacitor (SC) Resonant Resonat piezo Hybrid - SI / SC

Cytotoxic constituents from Brazilian red propolis and their structure-activity relationship.

PubMed

Li, Feng; Awale, Suresh; Tezuka, Yasuhiro; Kadota, Shigetoshi

2008-05-15

Several classes of flavonoids [flavanoids (1-10), flavonol (11), isoflavones (12-18), isoflavanones (19-22), isoflavans (23-26), chalcones (27-30), auronol (31), pterocarpans (32-37), 2-arylbenzofuran (38), and neoflavonoid (39)] and lignans (40-42) isolated from the MeOH extract of Brazilian red propolis were investigated for their cytotoxic activity against a panel of six different cancer cell lines including murine colon 26-L5 carcinoma, murine B16-BL6 melanoma, murine Lewis lung carcinoma, human lung A549 adenocarcinoma, human cervix HeLa adenocarcinoma, and human HT-1080 fibrosarcoma cell lines. Based on the observed results, structure-activity relationships were discussed. Among the tested compounds, 7-hydroxy-6-methoxyflavanone (3) exhibited the most potent activity against B16-BL6 (IC(50), 6.66microM), LLC (IC(50), 9.29microM), A549 (IC(50), 8.63microM), and HT-1080 (IC(50), 7.94microM) cancer cell lines, and mucronulatol (26) against LLC (IC(50), 8.38microM) and A549 (IC(50), 9.9microM) cancer cell lines. These activity data were comparable to those of the clinically used anticancer drugs, 5-fluorouracil and doxorubicin, against the tested cell lines, suggesting that 3 and 26 are the good candidates for future anticancer drug development.

Antitumor effect of cordycepin (3'-deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation.

PubMed

Nakamura, Kazuki; Yoshikawa, Noriko; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

2006-01-01

An attempt was made to elucidate the molecular targetfor the antitumor effects of cordycepin (3'-deoxyadenosine) using non-selective and selective adenosine A1, A2a, A2b and A3 receptor agonists and antagonists. Although adenosine and 2'-deoxyadenosine (up to 100 microM) had no effect, cordycepin showed remarkable inhibitory effects on the growth curves of B16-BL6 mouse melanoma (IC50= 39 microM) and mouse Lewis lung carcinoma (IC50 = 48 microM) cell lines in vitro. Among the adenosine receptor agonists and antagonists used (up to 100 microM), only 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective adenosine A3 receptor agonist, notably inhibited the growth of both mouse tumor cell lines (B16-BL6; IC50 = 5 microM, LLC; 14 microM). In addition, the tumor growth inhibitory effect of cordycepin was antagonized by 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191), a selective adenosine A3 receptor antagonist. These results suggest that cordycepin exerts inhibitory effects on the growth of mouse melanoma and lung carcinoma cells by stimulating adenosine A3 receptors on tumor cells.

MicroRNA inhibition fine-tunes and provides robustness to the restriction point switch of the cell cycle.

PubMed

Del Rosario, Ricardo C H; Damasco, Joseph Ray Clarence G; Aguda, Baltazar D

2016-09-09

The restriction point marks a switch in G1 from growth factor-dependent to growth factor-independent progression of the cell cycle. The proper regulation of this switch is important for normal cell processes; aberrations could result in a number of diseases such as cancer, neurodegenerative disorders, stroke and myocardial infarction. To further understand the regulation of the restriction point, we extended a mathematical model of the Rb-E2F pathway to include members of the microRNA cluster miR-17-92. Our mathematical analysis shows that microRNAs play an essential role in fine-tuning and providing robustness to the switch. We also demonstrate how microRNA regulation can steer cells in or out of cancer states.

MicroRNA inhibition fine-tunes and provides robustness to the restriction point switch of the cell cycle

PubMed Central

del Rosario, Ricardo C. H.; Damasco, Joseph Ray Clarence G.; Aguda, Baltazar D.

2016-01-01

The restriction point marks a switch in G1 from growth factor-dependent to growth factor-independent progression of the cell cycle. The proper regulation of this switch is important for normal cell processes; aberrations could result in a number of diseases such as cancer, neurodegenerative disorders, stroke and myocardial infarction. To further understand the regulation of the restriction point, we extended a mathematical model of the Rb-E2F pathway to include members of the microRNA cluster miR-17-92. Our mathematical analysis shows that microRNAs play an essential role in fine-tuning and providing robustness to the switch. We also demonstrate how microRNA regulation can steer cells in or out of cancer states. PMID:27610602

Determination of glucose in human stomach cancer cell extracts and single cells by capillary electrophoresis with a micro-biosensor.

PubMed

Wang, Xiaolei; Ma, Yanfang; Zhao, Man; Zhou, Minfeng; Xiao, Yan; Sun, Zifei; Tong, Lili

2016-10-21

Bioactive species in cells can provide information about signal transduction, cell function, and the effects of disease treatment. In this article, a novel micro-biosensor was fabricated to detect glucose in individual human stomach cancer cells (MGC80-3 cells) with capillary electrophoresis (CE). We fabricated the micro-biosensors by immobilizing a single-walled carbon nanotube-glucose oxidase (GOx)-glutaraldehyde (GA) bio-composite at the palladium nanoparticle (PdNPs) modified Pt electrode. The linear concentration of glucose ranged from 2.0μM to 1.0mM, with a detection limit of 0.5μM. Using this method, the mean amount of glucose in MGC80-3 cell extracts and in single cells was 20.0 fmol and 20±6 fmol (n=10), respectively. The micro-biosensor exhibited high sensitivity, stability, and a long operating life, which are likely due to the biocompatible environment provided by BSA and GA, and the adsorption and faster electron transfer of SWNTs and PdNPs to GOx. Copyright © 2016. Published by Elsevier B.V.

Real Time Monitoring of Temperature of a Micro Proton Exchange Membrane Fuel Cell

PubMed Central

Lee, Chi-Yuan; Lee, Shuo-Jen; Hu, Yuh-Chung; Shih, Wen-Pin; Fan, Wei-Yuan; Chuang, Chih-Wei

2009-01-01

Silicon micro-hole arrays (Si-MHA) were fabricated as a gas diffusion layer (GDL) in a micro fuel cell using the micro-electro-mechanical-systems (MEMS) fabrication technique. The resistance temperature detector (RTD) sensor was integrated with the GDL on a bipolar plate to measure the temperature inside the fuel cell. Experimental results demonstrate that temperature was generally linearly related to resistance and that accuracy and sensitivity were within 0.5 °C and 1.68×10−3/°C, respectively. The best experimental performance was 9.37 mW/cm2 at an H2/O2 dry gas flow rate of 30/30 SCCM. Fuel cell temperature during operation was 27 °C, as measured using thermocouples in contact with the backside of the electrode. Fuel cell operating temperature measured in situ was 30.5 °C. PMID:22573963

Thin concentrator photovoltaic module with micro-solar cells which are mounted by self-align method using surface tension of melted solder

NASA Astrophysics Data System (ADS)

Hayashi, Nobuhiko; Terauchi, Masaharu; Aya, Youichirou; Kanayama, Shutetsu; Nishitani, Hikaru; Nakagawa, Tohru; Takase, Michihiko

2017-09-01

We are developing a thin and lightweight CPV module using small size lens system made from poly methyl methacrylate (PMMA) with a short focal length and micro-solar cells to decrease the transporting and the installing costs of CPV systems. In order to achieve high conversion efficiency in CPV modules using micro-solar cells, the micro-solar cells need to be mounted accurately to the irradiated region of the concentrated sunlight. In this study, we have successfully developed self-align method thanks to the surface tension of the melted solder even utilizing commercially available surface-mounting technology (SMT). Solar cells were self-aligned to the specified positions of the circuit board by this self-align method with accuracy within ±10 µm. We actually fabricated CPV modules using this self-align method and demonstrated high conversion efficiency of our CPV module.

Cytotoxic effect of butein on human colon adenocarcinoma cell proliferation.

PubMed

Yit, C C; Das, N P

1994-07-15

Several classes of plant polyphenols namely, flavonoids, chalcones and coumarins exhibited varying degrees of inhibition on the cell proliferation of human colon adenocarcinoma cell line 220.1. At the highest concentration tested (100 microM), many of the chalcones showed > 100% growth inhibition and their order of potency was butein > 2'-hydroxychalcone > 2-hydroxychalcone > 2',6'-dihydroxy-4'-methoxychalcone > 2',4-dihydroxychalcone with IC50 values of 1.75, 6.2, 7.5, 17, 23 microM, respectively. Butein (the most potent chalcone) at 2 microM concentration inhibited the incorporation of 14C-labelled thymidine, uridine and leucine into the colon cancer cells whilst 5-fluorouracil (5-FU, a chemotherapeutic drug) at 50 microM concentration could significantly inhibit only the uridine incorporation. The mode of cytotoxic action of butein was different from 5-FU but may be similar to colchicine, a known HeLa cell inhibitor.

Mechanisms of Cdc42-mediated rat MSC differentiation on micro/nano-textured topography.

PubMed

Li, Guangwen; Song, Yanyan; Shi, Mengqi; Du, Yuanhong; Wang, Wei; Zhang, Yumei

2017-02-01

Micro/nano-textured titanium surface topography promotes osteoblast differentiation and the Wnt/β-catenin signaling pathway. However, the response of rat bone mesenchymal stem cells (MSCs) to micro/nano-textured topography, and the underlying mechanisms of its effects, are not well understood. We hypothesized that cell division cycle 42 protein (Cdc42), a key member of the Rho GTPases family, may regulate rat MSCs morphology and osteogenic differentiation by micro/nano-textured topography, and that crosstalk between Cdc42 and Wnt/β-catenin is the underlying mechanism. To confirm the hypothesis, we first tested rat MSCs' morphology, cytoskeleton, and osteogenic differentiation on micro/nano-textured topography. We then examined the cells' Wnt pathway and Cdc42 signaling activity. The results show that micro/nano-textured topography enhances MSCs' osteogenic differentiation. In addition, the cells' morphology and cytoskeletal reorganization were dramatically different on smooth surfaces and micropitted/nanotubular topography. Ligands of the canonical Wnt pathway, as well as accumulation of β-catenin in the nucleus, were up-regulated by micro/nano-textured topography. Cdc42 protein expression was markedly increased under these conditions; conversely, Cdc42 silencing significantly depressed the enhancement of MSCs osteogenic differentiation by micro/nano-textured topography. Moreover, Cdc42si attenuated p-GSK3β activation and resulted in β-catenin cytoplasmic degradation on the micro/nano-textured topography. Our results indicate that Cdc42 is a key modulator of rat MSCs morphology and cytoskeletal reorganization, and that crosstalk between Cdc42 and Wnt/β-catenin signaling though GSK3β regulates MSCs osteogenic differentiation by implant topographical cues. Topographical modification at micro- and nanoscale is widely applied to enhance the tissue integration properties of biomaterials. However, the response of bone mesenchymal stem cells (MSCs) to the micro/nano-textured topography and the underlying mechanisms are not well understood. This study shows that the micropitted/nanotubular hierarchical topography produced by etching and anodic oxidation treatment drives fusiform cell morphology, cytoskeletal reorganization as well as better MSCs osteogenic differentiation. The cross-talk between Cdc42 pathway and Wnt/β-catenin pathway though GSK3β modulates the osteoinductive effect of the micro/nano-textured topography on MSCs. This finding sheds light on a novel mechanism involved in micro/nano-textured surface-mediated MSCs osteogenic differentiation and is a major step in the development of new surface modifications aiming to accelerate and enhance the process of osseointegration. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

[Research Progress in Technology of Using Soil Micro-organisms to Generate Electricity and Its Potential Applications].

PubMed

Deng, Huan; Xue, Hong-jing; Jiang, Yun-bin; Zhong, Wen-hui

2015-10-01

Microbial fuel cells ( microbial fuel cells, MFCs) are devices in which micro-organisms convert chemical energy into electrical power. Soil has electrogenic bacteria and organic substrates, thus can generate electrical current in MFCs. Soil MFCs can be operated and applied to real-time and continuously monitor soil pollution, remove soil pollutants and to reduce methane emitted from flooded rice paddy, without energy consumption and the application of chemical reagents to the soil. Instead, the operation of soil MFCs generates small amount of electrical power. Therefore, soil MFCs are useful in the development of environment-friendly technology for monitoring and remediating soil pollution, which have potential value for applications in the domain of environmental science and engineering. However, much of advanced technology hasn't been applied into soil MFCs since the studies on soil MFCs was not started until recently. This paper summarized the research progress in related to soil MFCs combining with the frontier of MFCs technology, and brought forward the possible direction in studies on soil MFCs.

Betulinic Acid Targets YY1 and ErbB2 through Cannabinoid Receptor-Dependent Disruption of MicroRNA-27a:ZBTB10 in Breast Cancer

PubMed Central

Liu, Xinyi; Jutooru, Indira; Lei, Ping; Kim, KyoungHyun; Lee, Syng-ook; Brents, Lisa K.; Prather, Paul L.; Safe, Stephen

2016-01-01

Treatment of ErbB2-overexpressing BT474 and MDA-MB-453 breast cancer cells with 1 to 10 μmol/L betulinic acid inhibited cell growth, induced apoptosis, downregulated specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and decreased expression of ErbB2. Individual or combined knockdown of Sp1, Sp3, Sp4 by RNA interference also decreased expression of ErbB2 and this response was because of repression of YY1, an Sp-regulated gene. Betulinic acid–dependent repression of Sp1, Sp3, Sp4, and Sp-regulated genes was due, in part, to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a), which constitutively inhibits ZBTB10 expression, and we show for the first time that the effects of betulinic acid on the miR-27a:ZBTB10-Sp transcription factor axis were cannabinoid 1 (CB1) and CB2 receptor–dependent, thus identifying a new cellular target for this anticancer agent. PMID:22553354

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

NASA Astrophysics Data System (ADS)

Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

2018-05-01

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

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

PubMed Central

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

2010-01-01

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. PMID:20694048

Retinoic acid induction of calcium channel expression in human NT2N neurons.

PubMed

Gao, Z Y; Xu, G; Stwora-Wojczyk, M M; Matschinsky, F M; Lee, V M; Wolf, B A

1998-06-18

Ca2+ channel expression and regulation of intracellular Ca2+ homeostasis were studied during retinoic acid (RA)-induced differentiation of the human teratocarcinoma cell line Ntera 2/C1.D1 (NT2- cells) into NT2N neurons, a unique model of human neurons in culture. The cytosolic Ca2+ level of undifferentiated NT2- cells was low (75 +/- 5 nM) and stable under basal conditions, and it was only marginally decreased (by 9%) upon removal of extracellular Ca2+. After 10 microM RA treatment, NT2- cells were irreversibly differentiated into a phenotype of neuron-like NT2N cells. Cytosolic Ca2+ level of NT2N neurons was higher (106 +/- 14 nM) than that of NT2- cells and spontaneously fluctuated (0.208 +/- 0.038 transients/min) under basal conditions. Although K+ increased 86Rb fluxes in both NT2- cells and NT2N neurons, it only increased cytosolic Ca2+ level in NT2N neurons. The K+-induced increase in cytosolic Ca2+ in NT2N neurons was antagonized by 0.1-10 microM nifedipine or verapamil, 5 microM omega-CgTx GVIA, but not by 1 microM omega-agatoxin IVA, 1 microM omega-agatoxin TK, 1 microM FTX-3.3, or 100 microM Ni+ implicating L- and N-type voltage-dependent Ca2+ channels. In L- and N-type channels, but not in P- and Q-types, mRNAs were expressed in NT2N neurons as well as NT2- cells. Quantitative analysis of L- and N-type Ca2+ protein levels showed major differences between NT2- cells and NT2N neurons. In NT2- cells, N-type Ca2+ channels were undetectable while L-type channels levels were fivefold lower compared to NT2N neurons. Our findings show that L- and N-type channels are expressed during differentiation of NT2- cells into neurons, and that these voltage-dependent Ca2+ channels have a major role in regulating intracellular Ca2+ homeostasis and neuronal excitability. Copyright 1998 Academic Press.

From macro- to micro-single chamber solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Buergler, B. E.; Ochsner, M.; Vuillemin, S.; Gauckler, L. J.

Single chamber solid oxide fuel cells (SC-SOFCs) with interdigitating electrodes were prepared and operated in CH 4/air mixtures. Both electrodes (Ni-Ce 0.8Gd 0.2O 1.9 cermet and Sm 0.5Sr 0.5CoO 3- δ perovskite) were placed on the same side of a Ce 0.8Gd 0.1O 1.95 electrolyte disc. The separating gap between the electrodes was varied from 1.2 to 0.27 mm and finally down to 10 μm. Screen-printing was used for the preparation of the cells with a gap in the millimetre range, whereas micromolding in capillaries (MIMIC) was used for the preparation of the micro-SC-SOFCs. The prepared micro-SC-SOFCs consisted of an array of 19 individual cells that were connected in parallel having 100 μm wide electrodes. An open circuit voltage of 0.65-0.75 V was measured in flowing mixtures of methane and air. The maximum power density of 17 mW cm -2 was limited by the ohmic resistance of the long conduction paths along the thin electrodes to the active sites of the individual cells. The feasibility of the micro-cell was demonstrated by comparing the performance with the performance of the cells having feature sizes in the millimetre range. The cell resistance of micro-SC-SOFCs may be significantly reduced when connecting the cells in series using interconnections between anode and cathodes of adjacent cells.

Visualization of sporopollenin-containing pathogenic green micro-alga Prototheca wickerhamii by fluorescent in situ hybridization (FISH).

PubMed

Ueno, Ryohei

2009-04-01

Fluorescent in situ hybridization (FISH) using taxon-specific, rRNA-targeted oligonucleotide probes is one of the most powerful tools for the rapid identification of harmful microorganisms. However, eukaryotic algal cells do not always allow FISH probes to permeate over their cell walls. Members of the pathogenic micro-algal genus Prototheca are characterized by their distinctive cell-wall component, sporopollenin, an extremely tough biopolymer that resists acid and alkaline hydrolysis, enzyme attack, and acetolysis. To our knowledge, there has been no report of the successful permeation by the oligonucleotide probes over the cell walls of unicellular green micro-algae, which contain sporopollenin. The DNA probes passed through the cell wall of Prototheca wickerhamii after treating the algal cells with cetyltrimethylammonium bromide (CTAB). Most cells in the middle logarithmic growth phase culture fluoresced when hybridized with the rRNA-targeted universal probe for eukaryotes, though individual cells included in this culture differed in the level of cell-wall vulnerability to attack by the polysaccharide-degrading enzyme, thus reflecting the different stages of the life cycle. This is the first report regarding the visualization of sporopollenin-containing, green micro-algal cells by FISH.

VRP09 Reduction of Corneal Scarring Following Blast and Burn Injuries to Cornea Using siRNAs Targeting TGFb and CTGF

DTIC Science & Technology

2013-03-01

oligonucleotide-based drug approaches (better than ribozymes, antisense oligonucleotides ( ASO ), or microRNAs). (4) To accomplish these objectives, we...negative control scrambled ASO (designated NC). The combination of siRNAs T1 and R1 produced a knockdown of ~80% of TGFb1 protein in the conditioned...sequences (antisense oligonucleotides, ASOs ) into rabbit corneal cells and found that technique was very effective in delivering ASOs into the stroma and

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-beta, and H2O2.

PubMed

Lehmann, Petra; Rank, Petra; Hallfeldt, Klaus L J; Krebs, Bjarne; Gärtner, Roland

2006-08-01

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), as well as H2O2 and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-beta (5 ng/mL), or H2O2 (100 muM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. Asignificant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 microM), thyroidstimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 microM iodine, EGF (5 ng/mL) and TGF-beta (5 ng/mL), or H2O2 (100 microM) (p < 0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H2O2-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4 +/- 0.2 to 2.25 +/- 0.4 mU/microg DNA with 10 nMselenite and 2.6 + 0.4 mU/microg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-beta, EGF, iodide, and even H2O2.

Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway Regulated-Circulating microRNA

DTIC Science & Technology

2016-05-01

Award Number: W81XWH-11-1-0715 TITLE: Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway-Regulated Circulating microRNA PRINCIPAL...TITLE AND SUBTITLE Sa. CONTRACT NUMBER Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway- Regulated Circulating microRNA Sb. GRANT NUMBER...panel of diagnostic miRNAs that are measurable in serum and will be able to identify kidney cancer in its earliest stages. We hypothesized that serum

Emblica officinalis extract downregulates pro-angiogenic molecules via upregulation of cellular and exosomal miR-375 in human ovarian cancer cells

PubMed Central

De, Alok; Powers, Benjamin; De, Archana; Zhou, Jianping; Sharma, Siddarth; Van Veldhuizen, Peter; Bansal, Ajay; Sharma, Ramratan; Sharma, Mukut

2016-01-01

Ovarian cancer (OC) is highly resistant to current treatment strategies based on a combination of surgery, chemotherapy and radiation therapy. We have recently demonstrated the anti-neoplastic effect of Amla extract (Emblica officinalis, AE) on OC cells in vitro and in vivo. We hypothesized that AE attenuates growth of OC through microRNA (miR)-regulated mechanism(s). The inhibitory effect of AE on proliferation, migration and invasiveness (P≤0.001) of SKOV3 cells and >90% attenuation of tumor growth in a xenograft mouse model suggested multiple targets. RT-qPCR analysis of microRNAs associated with OC showed a >2,000-fold increase in the expression of miR-375 in AE-treated SKOV3 cells that was blocked by an exogenous miR-375 inhibitor (P≤0.001). AE also decreased the gene and protein expression of IGF1R, a target of miR-375 (P≤0.001), and SNAIL1 (P≤0.002), an EMT-associated transcription factor that represses E-cadherin expression (P≤0.003). AE increased E-cadherin expression (P≤0.001). Treatment of SKOV3 cells with AE resulted in increased miR-375 in exosomes in the medium (P≤0.01). Finally, AE significantly decreased the expression of IGF1R and SNAIL1 proteins during attenuation of SKOV3-derived xenograft tumor. Together, these results show that AE modulates cancer cells and the tumor microenvironment via activation of miR-375 and by targeting IGF1R and SNAIL1 in OC cells. PMID:27129171

Imaging trace element distributions in single organelles and subcellular features

NASA Astrophysics Data System (ADS)

Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

2016-02-01

The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

An Experimental Study on Micro Clinching of Metal Foils with Cutting by Laser Shock Forming.

PubMed

Wang, Xiao; Li, Cong; Ma, Youjuan; Shen, Zongbao; Sun, Xianqing; Sha, Chaofei; Gao, Shuai; Li, Liyin; Liu, Huixia

2016-07-13

This paper describes a novel technique for joining similar and dissimilar metal foils, namely micro clinching with cutting by laser shock forming. A series of experiments were conducted to study the deformation behavior of single layer material, during which many important process parameters were determined. The process window of the 1060 pure aluminum foils and annealed copper foils produced by micro clinching with cutting was analyzed. Moreover, similar material combination (annealed copper foils) and dissimilar material combination (1060 pure aluminum foils and 304 stainless steel foils) were successfully achieved. The effect of laser energy on the interlock and minimum thickness of upper foils was investigated. In addition, the mechanical strength of different material combinations joined by micro clinching with cutting was measured in single lap shearing tests. According to the achieved results, this novel technique is more suitable for material combinations where the upper foil is thicker than lower foil. With the increase of laser energy, the interlock increased while the minimum thickness of upper foil decreased gradually. The shear strength of 1060 pure aluminum foils and 304 stainless steel foils combination was three times as large as that of 1060 pure aluminum foils and annealed copper foils combination.

Resolving colocalization of bacteria and metal(loid)s on plant root surfaces by combining fluorescence in situ hybridization (FISH) with multiple-energy micro-focused X-ray fluorescence (ME μXRF).

PubMed

Honeker, Linnea K; Root, Robert A; Chorover, Jon; Maier, Raina M

2016-12-01

Metal(loid)-contamination of the environment due to anthropogenic activities is a global problem. Understanding the fate of contaminants requires elucidation of biotic and abiotic factors that influence metal(loid) speciation from molecular to field scales. Improved methods are needed to assess micro-scale processes, such as those occurring at biogeochemical interfaces between plant tissues, microbial cells, and metal(loid)s. Here we present an advanced method that combines fluorescence in situ hybridization (FISH) with synchrotron-based multiple-energy micro-focused X-ray fluorescence microprobe imaging (ME μXRF) to examine colocalization of bacteria and metal(loid)s on root surfaces of plants used to phytostabilize metalliferous mine tailings. Bacteria were visualized on a small root section using SytoBC nucleic acid stain and FISH probes targeting the domain Bacteria and a specific group (Alphaproteobacteria, Gammaproteobacteria, or Actinobacteria). The same root region was then analyzed for elemental distribution and metal(loid) speciation of As and Fe using ME μXRF. The FISH and ME μXRF images were aligned using ImageJ software to correlate microbiological and geochemical results. Results from quantitative analysis of colocalization show a significantly higher fraction of As colocalized with Fe-oxide plaques on the root surfaces (fraction of overlap 0.49±0.19) than to bacteria (0.072±0.052) (p<0.05). Of the bacteria that colocalized with metal(loid)s, Actinobacteria, known for their metal tolerance, had a higher correlation with both As and Fe than Alphaproteobacteria or Gammaproteobacteria. This method demonstrates how coupling these micro-techniques can expand our understanding of micro-scale interactions between roots, metal(loid)s and microbes, information that should lead to improved mechanistic models of metal(loid) speciation and fate. Copyright © 2016 Elsevier B.V. All rights reserved.

Two microRNA panels to discriminate three subtypes of lung carcinoma in bronchial brushing specimens.

PubMed

Huang, Wei; Hu, Jie; Yang, Da-wei; Fan, Xin-ting; Jin, Yi; Hou, Ying-yong; Wang, Ji-ping; Yuan, Yun-feng; Tan, Yun-shan; Zhu, Xiong-Zeng; Bai, Chun-xue; Wu, Ying; Zhu, Hong-guang; Lu, Shao-hua

2012-12-01

Effective treatment for lung cancer requires accuracy in subclassification of carcinoma subtypes. To identify microRNAs in bronchial brushing specimens for discriminating small cell lung cancer (SCLC) from non-small cell lung cancer (NSCLC) and for further differentiating squamous cell carcinoma (SQ) from adenocarcinoma (AC). Microarrays were used to screen 723 microRNAs in laser-captured, microdissected cancer cells from 82 snap-frozen surgical lung specimens. Quantitative reverse-transcriptase polymerase chain reaction was performed on 153 macrodissected formalin-fixed, paraffin-embedded (FFPE) surgical lung specimens to evaluate seven microRNA candidates discovered from microarrays. Two microRNA panels were constructed on the basis of a training cohort (n = 85) and validated using an independent cohort (n = 68). The microRNA panels were applied as differentiators of SCLC from NSCLC and of SQ from AC in 207 bronchial brushing specimens. Two microRNA panels yielded high diagnostic accuracy in discriminating SCLC from NSCLC (miR-29a and miR-375; area under the curve [AUC], 0.991 and 0.982 for training and validation data set, respectively) and in differentiating SQ from AC (miR-205 and miR-34a; AUC, 0.977 and 0.982 for training and validation data set, respectively) in FFPE surgical lung specimens. Moreover, the microRNA panels accurately differentiated SCLC from NSCLC (AUC, 0.947) and SQ from AC (AUC, 0.962) in bronchial brushing specimens. We found two microRNA panels that accurately discriminated between the three subtypes of lung carcinoma in bronchial brushing specimens. The identified microRNA panels may have considerable clinical value in differential diagnosis and optimizing treatment strategies based on lung cancer subtypes.

Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation

PubMed Central

Alsaweed, Mohammed; Hepworth, Anna R.; Lefèvre, Christophe; Hartmann, Peter E.; Geddes, Donna T.

2015-01-01

ABSTRACT MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing, and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises three main fractions: cells, skim milk, and lipids. Typically, the skim milk fraction has been measured in isolation despite evidence that the lipid fraction may contain more microRNA. This study aimed to standardize isolation of microRNA and total RNA from all three fractions of human milk to determine the most appropriate sampling and analysis procedure for future studies. Three different methods from eight commercially available kits were tested for their efficacy in extracting total RNA and microRNA from the lipid, skim, and cell fractions of human milk. Each fraction yielded different concentrations of RNA and microRNA, with the highest quantities found in the cell and lipid fractions, and the lowest in skim milk. The column‐based phenol‐free method was the most efficient extraction method for all three milk fractions. Two microRNAs were expressed and validated in the three milk fractions by qPCR using the three recommended extraction kits for each fraction. High expression levels were identified in the skim and lipid milk factions for these microRNAs. These results suggest that careful consideration of both the human milk sample preparation and extraction protocols should be made prior to embarking upon research in this area. J. Cell. Biochem. 116: 2397–2407, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. PMID:25925799

The thermomechanical stability of micro-solid oxide fuel cells fabricated on anodized aluminum oxide membranes

NASA Astrophysics Data System (ADS)

Kwon, Chang-Woo; Lee, Jae-Il; Kim, Ki-Bum; Lee, Hae-Weon; Lee, Jong-Ho; Son, Ji-Won

2012-07-01

The thermomechanical stability of micro-solid oxide fuel cells (micro-SOFCs) fabricated on an anodized aluminum oxide (AAO) membrane template is investigated. The full structure consists of the following layers: AAO membrane (600 nm)/Pt anode/YSZ electrolyte (900 nm)/porous Pt cathode. The utilization of a 600-nm-thick AAO membrane significantly improves the thermomechanical stability due to its well-known honeycomb-shaped nanopore structure. Moreover, the Pt anode layer deposited in between the AAO membrane and the YSZ electrolyte preserves its integrity in terms of maintaining the triple-phase boundary (TPB) and electrical conductivity during high-temperature operation. Both of these results guarantee thermomechanical stability of the micro-SOFC and extend the cell lifetime, which is one of the most critical issues in the fabrication of freestanding membrane-type micro-SOFCs.

The Role of Magnesium Ion Substituted Biphasic Calcium Phosphate Spherical Micro-Scaffolds in Osteogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

PubMed

Kim, Dong-Hyun; Shin, Keun-Koo; Jung, Jin Sup; Chun, Ho Hwan; Park, Seong Soo; Lee, Jong Kook; Park, Hong-Chae; Yoon, Seog-Young

2015-08-01

This study was investigated the role of magnesium (Mg2+) ion substituted biphasic calcium phosphate (Mg-BCP) spherical micro-scaffolds in osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs). Mg-BCP micro-scaffolds with spherical morphology were successfully prepared using in situ co-precipitation and spray drying atomization process. The in vitro cell proliferation and differentiation of hAT-MSCs were determined up to day 14. After in vitro biological tests, Mg-BCP micro-scaffolds with hAT-MSCs showed more enhanced osteogenicity than pure hAT-MSCs as control group by unique biodegradation of TCP phase and influence of substituted Mg2+ ion in biphasic nanostructure. Therefore, these results suggest that Mg-BCP micro-scaffolds promote osteogenic differentiation of hAT-MSCs.

Viability of a human melanoma cell after single and combined treatment with fotemustine, dacarbazine, and proton irradiation.

PubMed

Petrović, Ivan M; Korićanac, Lela B; Todorović, Danijela V; Ristić-Fira, Aleksandra M; Valastro, Lucia M; Privitera, Giuseppe; Cuttone, Giacomo

2007-01-01

Viability of human HTB140 melanoma cells after being exposed to fotemustine (FM) and dacarbazine (DTIC) as well as to proton irradiation was studied. Effects of 100 and 250 microM drugs were assessed after incubation of 6, 24, 48, 72, and 96 h. Irradiations were performed with 62 MeV therapeutic protons, delivering to the cell monolayer single doses of 2, 4, 8, 12, and 16 Gy. Viability was evaluated 7 days after irradiation. Inactivation level was estimated using microtetrasolium (MTT) and sulforhodamine B (SRB) assays. Combined effects of each drug and protons, were carried out using the same drug concentrations. Proton doses applied were those used in therapy, that is, 12 and 16 Gy. With the increase of drug concentration or irradiation dose, level of cell inactivation reached approximately 60%, 48 h after drug treatment or 7 days after irradiation at 16 Gy. Considering the rate of drug concentrations used, as well as the level of doses applied, it appears that HTB140 cells are more resistant to proton irradiation than to alkylating agents tested. The combined treatment with FM or DTIC and protons did not show significant changes of cell viability as compared to the effects of single agents. Since the time point for measuring cumulative effects of drug and irradiation was 48 h post irradiation, it seems that the obtained level of viability could be attributed primarily to the effects of drugs.

Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA).

PubMed

Hannafon, Bethany N; Carpenter, Karla J; Berry, William L; Janknecht, Ralf; Dooley, William C; Ding, Wei-Qun

2015-07-16

Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA's anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA's anticancer action. Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA's anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA's anticancer action, further supporting its use in cancer therapy.

A Chelator-Free Multifunctional [64Cu]-CuS Nanoparticle Platform for Simultaneous Micro-PET/CT Imaging and Photothermal Ablation Therapy

PubMed Central

Zhou, Min; Zhang, Rui; Huang, Miao; Lu, Wei; Song, Shaoli; Melancon, Marites P.; Tian, Mei; Liang, Dong; Li, Chun

2010-01-01

We synthesized and evaluated a novel class of chelator-free [64Cu]-CuS nanoparticles (NPs) suitable for both PET imaging and as photothermal coupling agents for photothermal ablation. [64Cu]-CuS NPs were simple to make, possessed excellent stability, and allowed robust noninvasive micro-PET imaging. Furthermore, CuS NPs displayed strong absorption in the near-infrared (NIR) region (peak 930 nm), passive targeting prefers the tumor site, and mediated ablation of U87 tumor cells upon exposure to NIR light both in vitro and in vivo after either intratumoral or intravenous injection. The combination of small diameter (~11 nm diameter), strong NIR absorption, and integration of 64Cu as a structural component makes [64Cu]-CuS NPs ideally suited for multifunctional molecular imaging and therapy. PMID:20942456

Use of self-actuating and self-sensing cantilevers for imaging biological samples in fluid

PubMed Central

Barbero, R J; Deutschinger, A; Todorov, V; Gray, D S; Belcher, A M; Rangelow, I W; Youcef-Toumi, K

2014-01-01

In this paper, we present a detailed investigation into the suitability of atomic force microscopy (AFM) cantilevers with integrated deflection sensor and micro-actuator for imaging of soft biological samples in fluid. The Si cantilevers are actuated using a micro-heater at the bottom end of the cantilever. Sensing is achieved through p-doped resistors connected in a Wheatstone bridge. We investigated the influence of the water on the cantilever dynamics, the actuation and the sensing mechanisms, as well as the crosstalk between sensing and actuation. Successful imaging of yeast cells in water using the integrated sensor and actuator shows the potential of the combination of this actuation and sensing method. This constitutes a major step towards the automation and miniaturization required to establish AFM in routine biomedical diagnostics and in vivo applications. PMID:19801750

Alpha-, gamma- and delta-tocopherols reduce inflammatory angiogenesis in human microvascular endothelial cells.

PubMed

Wells, Shannon R; Jennings, Merilyn H; Rome, Courtney; Hadjivassiliou, Vicky; Papas, Konstantinos A; Alexander, Jonathon S

2010-07-01

Vitamin E, a micronutrient (comprising alpha-, beta-, gamma- and delta-tocopherols, alpha-, beta-, gamma- and delta-tocotrienols), has documented antioxidant and non-antioxidant effects, some of which inhibit inflammation and angiogenesis. We compared the abilities of alpha-, gamma- and delta-tocopherols to regulate human blood cytotoxicity (BEC) and lymphatic endothelial cytotoxicity (LEC), proliferation, invasiveness, permeability, capillary formation and suppression of TNF-alpha-induced VCAM-1 as in vitro models of inflammatory angiogenesis. alpha-, gamma- and delta-tocopherols were not toxic to either cell type up to 40 microM. In BEC, confluent cell density was decreased by all concentrations of delta- and gamma-tocopherol (10-40 microM) but not by alpha-tocopherol. LEC showed no change in cell density in response to tocopherols. delta-Tocopherol (40 microM), but not other isomers, decreased BEC invasiveness. In LEC, all doses of gamma-tocopherol, as well as the highest dose of alpha-tocopherol (40 microM), decreased cell invasiveness. delta-Tocopherol had no effect on LEC invasiveness at any molarity. delta-Tocopherol dose dependently increased cell permeability at 48 h in BEC and LEC; alpha- and gamma-tocopherols showed slight effects. Capillary tube formation was decreased by high dose (40 microM) concentrations of alpha-, gamma- and delta-tocopherol, but showed no effects with smaller doses (10-20 microM) in BEC. gamma-Tocopherol (10-20 microM) and alpha-tocopherol (10 microM), but not delta-tocopherol, increased LEC capillary tube formation. Lastly, in BEC, alpha-, gamma- and delta-tocopherol each dose-dependently reduced TNF-alpha-induced expression of VCAM-1. In LEC, there was no significant change to TNF-alpha-induced VCAM-1 expression with any concentration of alpha-, gamma- or delta-tocopherol. These data demonstrate that physiological levels (0-40 microM) of alpha-, gamma- and delta-tocopherols are nontoxic and dietary tocopherols, especially delta-tocopherol, can limit several BEC and LEC endothelial behaviors associated with angiogenesis. Tocopherols may therefore represent important nutrient-signals that limit cell behaviors related to inflammation/angiogenesis, which when deficient, may predispose individuals to risks associated with elevated angiogenesis such as inflammation and cancer; further differences seen from the tocopherols may be due to their blood or lymphatic cell origin. (c) 2010 Elsevier Inc. All rights reserved.

Modeling of combined effects of citral, linalool and beta-pinene used against Saccharomyces cerevisiae in citrus-based beverages subjected to a mild heat treatment.

PubMed

Belletti, Nicoletta; Kamdem, Sylvain Sado; Tabanelli, Giulia; Lanciotti, Rosalba; Gardini, Fausto

2010-01-01

The aim of this work was to evaluate the antimicrobial activity of three terpenes (citral, linalool and beta-pinene), in combination with a mild heat treatment (55 degrees C, 15 min). The study has been carried out on an orange based soft drink inoculated using a wild strain of Saccharomyces cerevisiae. The results, expressed as growth/no-growth data, were analyzed with the logistic regression. A model comprising only of significant individual parameters (p < or = 0.05) and describing the relationships between terpene concentrations and the probability of having stable beverages was obtained. When citral and beta-pinene were combined, the citral concentration required to achieve a 50% probability of having stable bottles (P=0.5) dropped from 100.9 microL/L in the absence of beta-pinene to 49.3 microL/L in the presence of 20 microL/L of beta-pinene. The mixture of citral and linalool was less effective, in fact, the same probability (P=0.5) was obtained combining 60 microL/L of linalool with 35.1 microL/L of citral. The addition of 20 microL/L of linalool and beta-pinene reinforced citral bioactivity and the concentration of citral needed to reach P=0.5 fell from 100.9 microL/L in the presence of citral alone to 42.0 microL/L. The presence of both linalool and beta-pinene at a concentration of 40 or 60 microL/L in the absence of citral led to a lower spoilage probability (P=0.58 and P=0.93, respectively). It can be concluded that the antimicrobial potential of the three terpenes alone can be strengthened combining appropriate concentrations of each of them. This study confirmed also the potentiating effect of a mild temperature treatment on the antimicrobial efficacy of the molecules. Neither the thermal treatment alone nor the presence of the terpenes at their maximum concentrations (without thermal treatment) were able to guarantee the microbial stability of the beverages. 2009 Elsevier B.V. All rights reserved.

A novel method for in-situ monitoring of local voltage, temperature and humidity distributions in fuel cells using flexible multi-functional micro sensors.

PubMed

Lee, Chi-Yuan; Fan, Wei-Yuan; Chang, Chih-Ping

2011-01-01

In this investigation, micro voltage, temperature and humidity sensors were fabricated and integrated for the first time on a stainless steel foil using micro-electro-mechanical systems (MEMS). These flexible multi-functional micro sensors have the advantages of high temperature resistance, flexibility, smallness, high sensitivity and precision of location. They were embedded in a proton exchange membrane fuel cell (PEMFC) and used to simultaneously measure variations in the inner voltage, temperature and humidity. The accuracy and reproducibility of the calibrated results obtained using the proposed micro sensors is excellent. The experimental results indicate that, at high current density and 100%RH or 75%RH, the relative humidity midstream and downstream saturates due to severe flooding. The performance of the PEM fuel cell can be stabilized using home-made flexible multi-functional micro sensors by the in-situ monitoring of local voltage, temperature and humidity distributions within it.

A Novel Method for In-Situ Monitoring of Local Voltage, Temperature and Humidity Distributions in Fuel Cells Using Flexible Multi-Functional Micro Sensors

PubMed Central

Lee, Chi-Yuan; Fan, Wei-Yuan; Chang, Chih-Ping

2011-01-01

In this investigation, micro voltage, temperature and humidity sensors were fabricated and integrated for the first time on a stainless steel foil using micro-electro-mechanical systems (MEMS). These flexible multi-functional micro sensors have the advantages of high temperature resistance, flexibility, smallness, high sensitivity and precision of location. They were embedded in a proton exchange membrane fuel cell (PEMFC) and used to simultaneously measure variations in the inner voltage, temperature and humidity. The accuracy and reproducibility of the calibrated results obtained using the proposed micro sensors is excellent. The experimental results indicate that, at high current density and 100%RH or 75%RH, the relative humidity midstream and downstream saturates due to severe flooding. The performance of the PEM fuel cell can be stabilized using home-made flexible multi-functional micro sensors by the in-situ monitoring of local voltage, temperature and humidity distributions within it. PMID:22319361

Targeting Metabolic Remodeling in Triple Negative Breast Cancer in a Murine Model

PubMed Central

García-Castillo, Verónica; López-Urrutia, Eduardo; Villanueva-Sánchez, Octavio; Ávila-Rodríguez, Miguel Á.; Zentella-Dehesa, Alejandro; Cortés-González, Carlo; López-Camarillo, César; Jacobo-Herrera, Nadia J; Pérez-Plasencia, Carlos

2017-01-01

Background: Chemotherapy is the backbone of systemic treatment for triple negative breast cancer (TNBC), which is one of the most relevant breast cancers molecular types due to the ability of tumor cells to develop drug resistance, highlighting the urgent need to design newer and safer drug combinations for treatment. In this context, to overcome tumor cell drug resistance, we employed a novel combinatorial treatment including Doxorubicin, Metformin, and Sodium Oxamate (DoxMetOx). Such pharmacological combination targets indispensable hallmarks of cancer-related to aerobic glycolysis and DNA synthesis. Materials and Methods: Thirty-five female nude mice were transplanted subcutaneously with MDA-MB-231 triple negative human cancer cell line. Once tumors were visible, mice were treated with doxorubicin, metformin, oxamate or all possible pharmacologic combinations. Treatments were administered daily for 15 days and tumors were measured by calipers every day. MicroPET images were taken in three different occasions, basal state, in the middle of the treatment, and at the end of treatment. Western blot analyses, qRT-PCR, flow cytometry, and cytotoxicity assays were performed to elucidate the mechanism of cell death promoted by the drugs in vitro. Results: In this work we assessed the proof of concept of metabolic correction in solid tumors as an effective drug treatment; hence, mice bearing tumors treated with the DoxMetOx therapy showed a complete inhibition of the tumor mass growing in 15 days of treatment depicted by the micro PET images. In vitro studies displayed that the three drugs together act by inhibiting both, mTOR-phosphorylation and expression of LDH-A gene, promoting apoptosis via dependent on the caspase-3 pathway, accompanied by cleavage of PARP. Moreover, induction of autophagy process was observed by the accumulation of LC3-II, a primordial protein implicated in the conformation and elongation of the autophagolysosome. Conclusions: The lack of effective drugs to inhibit TNBC growth is the main cause of therapy failure and tumor relapse. We have showed that targeting crucial molecular pathways in cancer by the combination of Doxorubicin, Metformin, and Oxamate resulted as an efficient and rapid tumor growth inhibitor in a triple negative xenograft model. Our findings are promising for patients diagnosed with TNBC tumors, for which unfortunately there are no reliable drug therapies. PMID:28243322

Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

DTIC Science & Technology

2014-09-01

Watters, K. M., Das, S., Bryan, K., Bernas, T., Prehn , J. H., and Stallings, R. L. (2011) MicroRNAs 10a and 10b are potent inducers of neuroblastoma...Cell Biol 2009;29(19):5290-5305. 30. Foley NH, Bray I, Watters KM, Das S, Bryan K, Bernas T, Prehn JH, Stallings RL. MicroRNAs 10a and 10b are potent

Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels.

PubMed

Pissarek, M; Garcia de Arriba, S; Schäfer, M; Sieler, D; Nieber, K; Illes, P

1998-10-01

In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95%O2-5%CO2 with 95%N2-5%CO2) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K(ATP)) channels, diazoxide (3-300 microM). The effect of 30 but not 300 microM diazoxide was reversed by washout. Tolbutamide (300 microM), an antagonist of K(ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 microM) on the HD. Neither diazoxide (3-300 microM) nor tolbutamide (300 microM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 microM) or tolbutamide (300 microM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between -130 and -50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 microM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri- to diphosphate ratios progressively declined for ATP/ADP and GTP/GDP, but not for UTP/UDP when the duration of hypoxia was increased from 5 to 30 min. Hence, the rapid fall in the ratios of nucleoside tri- to diphosphates without the induction of a potassium current failed to indicate an allosteric regulation of a plasmalemmal K(ATP) channel by purine and pyrimidine nucleotides. Diazoxide had no effect on neocortical pyramidal neurons and was effective only in combination with a hypoxic stimulus; it is suggested that both plasmalemmal and mitochondrial K(ATP) channels are involved under these conditions. The hypoxic depolarization may be due to blockade of K+,Na+-ATPase by limitation of energy supplying substrate.

Hybrid 3D-2D printing for bone scaffolds fabrication

NASA Astrophysics Data System (ADS)

Seleznev, V. A.; Prinz, V. Ya

2017-02-01

It is a well-known fact that bone scaffold topography on micro- and nanometer scale influences the cellular behavior. Nano-scale surface modification of scaffolds allows the modulation of biological activity for enhanced cell differentiation. To date, there has been only a limited success in printing scaffolds with micro- and nano-scale features exposed on the surface. To improve on the currently available imperfect technologies, in our paper we introduce new hybrid technologies based on a combination of 2D (nano imprint) and 3D printing methods. The first method is based on using light projection 3D printing and simultaneous 2D nanostructuring of each of the layers during the formation of the 3D structure. The second method is based on the sequential integration of preliminarily created 2D nanostructured films into a 3D printed structure. The capabilities of the developed hybrid technologies are demonstrated with the example of forming 3D bone scaffolds. The proposed technologies can be used to fabricate complex 3D micro- and nanostructured products for various fields.

Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility.

PubMed

Liu, Hao; Chen, Wei; Zhi, Xiao; Chen, En-Jiang; Wei, Tao; Zhang, Jian; Shen, Jian; Hu, Li-Qiang; Zhao, Bin; Feng, Xin-Hua; Bai, Xue-Li; Liang, Ting-Bo

2018-05-22

Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells.

Accelerated detection of viral particles by combining AC electric field effects and micro-Raman spectroscopy.

PubMed

Tomkins, Matthew Robert; Liao, David Shiqi; Docoslis, Aristides

2015-01-08

A detection method that combines electric field-assisted virus capture on antibody-decorated surfaces with the "fingerprinting" capabilities of micro-Raman spectroscopy is demonstrated for the case of M13 virus in water. The proof-of-principle surface mapping of model bioparticles (protein coated polystyrene spheres) captured by an AC electric field between planar microelectrodes is presented with a methodology for analyzing the resulting spectra by comparing relative peak intensities. The same principle is applied to dielectrophoretically captured M13 phage particles whose presence is indirectly confirmed with micro-Raman spectroscopy using NeutrAvidin-Cy3 as a labeling molecule. It is concluded that the combination of electrokinetically driven virus sampling and micro-Raman based signal transduction provides a promising approach for time-efficient and in situ detection of viruses.

Accelerated Detection of Viral Particles by Combining AC Electric Field Effects and Micro-Raman Spectroscopy

PubMed Central

Tomkins, Matthew Robert; Liao, David Shiqi; Docoslis, Aristides

2015-01-01

A detection method that combines electric field-assisted virus capture on antibody-decorated surfaces with the “fingerprinting” capabilities of micro-Raman spectroscopy is demonstrated for the case of M13 virus in water. The proof-of-principle surface mapping of model bioparticles (protein coated polystyrene spheres) captured by an AC electric field between planar microelectrodes is presented with a methodology for analyzing the resulting spectra by comparing relative peak intensities. The same principle is applied to dielectrophoretically captured M13 phage particles whose presence is indirectly confirmed with micro-Raman spectroscopy using NeutrAvidin-Cy3 as a labeling molecule. It is concluded that the combination of electrokinetically driven virus sampling and micro-Raman based signal transduction provides a promising approach for time-efficient and in situ detection of viruses. PMID:25580902

Development of optics with micro-LED arrays for improved opto-electronic neural stimulation

NASA Astrophysics Data System (ADS)

Chaudet, Lionel; Neil, Mark; Degenaar, Patrick; Mehran, Kamyar; Berlinguer-Palmini, Rolando; Corbet, Brian; Maaskant, Pleun; Rogerson, David; Lanigan, Peter; Bamberg, Ernst; Roska, Botond

2013-03-01

The breakthrough discovery of a nanoscale optically gated ion channel protein, Channelrhodopsin 2 (ChR2), and its combination with a genetically expressed ion pump, Halorhodopsin, allowed the direct stimulation and inhibition of individual action potentials with light alone. This work reports developments of ultra-bright elec­ tronically controlled optical array sources with enhanced light gated ion channels and pumps for use in systems to further our understanding of both brain and visual function. This work is undertaken as part of the European project, OptoNeuro. Micro-LED arrays permit spatio-temporal control of neuron stimulation on sub-millisecond timescales. However they are disadvantaged by their broad spatial light emission distribution and low fill factor. We present the design and implementation of a projection and micro-optics system for use with a micro-LED array consisting of a 16x16 matrix of 25 μm diameter micro-LEDs with 150 μm centre-to-centre spacing and an emission spectrum centred at 470 nm overlapping the peak sensitivity of ChR2 and its testing on biological samples. The projection system images the micro-LED array onto micro-optics to improve the fill-factor from ~2% to more than 78% by capturing a larger fraction of the LED emission and directing it correctly to the sample plane. This approach allows low fill factor arrays to be used effectively, which in turn has benefits in terms of thermal management and electrical drive from CMOS backplane electronics. The entire projection system is integrated into a microscope prototype to provide stimulation spots at the same size as the neuron cell body (μ10 pm).

Electrochemical impedance spectroscopy analysis of a thin polymer film-based micro-direct methanol fuel cell

NASA Astrophysics Data System (ADS)

Schulz, Tobias; Weinmüller, Christian; Nabavi, Majid; Poulikakos, Dimos

A single cell micro-direct methanol fuel cell (micro-DMFC) was investigated using electrochemical impedance spectroscopy. The electrodes consisted of thin, flexible polymer (SU8) film microchannel structures fabricated in-house using microfabrication techniques. AC impedance spectroscopy was used to separate contributions to the overall cell polarization from the anode, cathode and membrane. A clear distinction between the different electrochemical phenomena occurring in the micro-DMFC, especially the distinction between double layer charging and Faradaic reactions was shown. The effect of fuel flow rate, temperature, and anode flow channel structure on the impedance of the electrode reactions and membrane/electrode double layer charging were investigated. Analysis of impedance data revealed that the performance of the test cell was largely limited by the presence of intermediate carbon monoxide in the anode reaction. Higher temperatures increase cell performance by enabling intermediate CO to be oxidized at much higher rates. The results also revealed that serpentine anode flow microchannels show a lower tendency to intermediate CO coverage and a more stable cell behavior than parallel microchannels.

Micro-masters of glioblastoma biology and therapy: increasingly recognized roles for microRNAs.

PubMed

Floyd, Desiree; Purow, Benjamin

2014-05-01

MicroRNAs are small noncoding RNAs encoded in eukaryotic genomes that have been found to play critical roles in most biological processes, including cancer. This is true for glioblastoma, the most common and lethal primary brain tumor, for which microRNAs have been shown to strongly influence cell viability, stem cell characteristics, invasiveness, angiogenesis, metabolism, and immune evasion. Developing microRNAs as prognostic markers or as therapeutic agents is showing increasing promise and has potential to reach the clinic in the next several years. This succinct review summarizes current progress and future directions in this exciting and steadily expanding field.

Micro-7 BioCell Habitat Fixation Operations

NASA Image and Video Library

2014-04-25

ISS039-E-015593 (25 April 2014) --- In the Harmony node of the Earth-orbiting International Space Station, NASA astronaut Rick Mastracchio, Expedition 39 flight engineer, works with the Micro-7 BioCell habitat.

Micro-7 BioCell Habitat Fixation Operations

NASA Image and Video Library

2014-04-25

ISS039-E-015646 (25 April 2014) --- In the Harmony node of the Earth-orbiting International Space Station, NASA astronaut Rick Mastracchio, Expedition 39 flight engineer, works with the Micro-7 BioCell habitat.

Micro- and nanoengineering for stem cell biology: the promise with a caution.

PubMed

Kshitiz; Kim, Deok-Ho; Beebe, David J; Levchenko, Andre

2011-08-01

Current techniques used in stem cell research only crudely mimic the physiological complexity of the stem cell niches. Recent advances in the field of micro- and nanoengineering have brought an array of in vitro cell culture models that have enabled development of novel, highly precise and standardized tools that capture physiological details in a single platform, with greater control, consistency, and throughput. In this review, we describe the micro- and nanotechnology-driven modern toolkit for stem cell biologists to design novel experiments in more physiological microenvironments with increased precision and standardization, and caution them against potential challenges that the modern technologies might present. Copyright © 2011 Elsevier Ltd. All rights reserved.

Interleukin-5 regulates genes involved in B-cell terminal maturation.

PubMed

Horikawa, Keisuke; Takatsu, Kiyoshi

2006-08-01

Interleukin (IL)-5 induces CD38-activated splenic B cells to differentiate into immunoglobulin M-secreting cells and undergo micro to gamma 1 class switch recombination (CSR) at the DNA level, resulting in immunoglobulin G1 (IgG1) production. Interestingly, IL-4, a well-known IgG1-inducing factor does not induce immunoglobulin production or micro to gamma 1 CSR in CD38-activated B cells. In the present study, we implemented complementary DNA microarrays to investigate the contribution of IL-5-induced gene expression in CD38-stimulated B cells to immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR. IL-5 and IL-4 stimulation of CD38-activated B cells induced the expression of 418 and 289 genes, respectively, that consisted of several clusters. Surprisingly, IL-5-inducible 78 genes were redundantly regulated by IL-4. IL-5 and IL-4 also suppressed the gene expression of 319 and 325 genes, respectively, 97 of which were overlapped. Genes critically regulated by IL-5 include immunoglobulin-related genes such as J chain and immunoglobulinkappa, and genes involved in B-cell maturation such as BCL6, activation-induced cytidine deaminase (Aid) and B lymphocyte-induced maturation protein-1 (Blimp-1) and tend to be induced slowly after IL-5 stimulation. Intriguingly, among genes, the retroviral induction of Blimp-1 and Aid in CD38-activated B cells could induce IL-4-dependent maturation to Syndecan-1+ antibody-secreting cells and micro to gamma 1 CSR, respectively, in CD38-activated B cells. Taken together, preferential Aid and Blimp-1 expression plays a critical role in IL-5-induced immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR in CD38-activated B cells.

Wafer integrated micro-scale concentrating photovoltaics

NASA Astrophysics Data System (ADS)

Gu, Tian; Li, Duanhui; Li, Lan; Jared, Bradley; Keeler, Gordon; Miller, Bill; Sweatt, William; Paap, Scott; Saavedra, Michael; Das, Ujjwal; Hegedus, Steve; Tauke-Pedretti, Anna; Hu, Juejun

2017-09-01

Recent development of a novel micro-scale PV/CPV technology is presented. The Wafer Integrated Micro-scale PV approach (WPV) seamlessly integrates multijunction micro-cells with a multi-functional silicon platform that provides optical micro-concentration, hybrid photovoltaic, and mechanical micro-assembly. The wafer-embedded micro-concentrating elements is shown to considerably improve the concentration-acceptance-angle product, potentially leading to dramatically reduced module materials and fabrication costs, sufficient angular tolerance for low-cost trackers, and an ultra-compact optical architecture, which makes the WPV module compatible with commercial flat panel infrastructures. The PV/CPV hybrid architecture further allows the collection of both direct and diffuse sunlight, thus extending the geographic and market domains for cost-effective PV system deployment. The WPV approach can potentially benefits from both the high performance of multijunction cells and the low cost of flat plate Si PV systems.

alpha 4 beta 2 subunit combination specific pharmacology of neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells.

PubMed

Zwart, R; Abraham, D; Oortgiesen, M; Vijverberg, H P

1994-08-22

Pharmacological characteristics of native neuronal nicotinic acetylcholine receptor-mediated ion currents in mouse N1E-115 neuroblastoma cells have been investigated by superfusion of voltage clamped cells with known concentrations of the agonists acetylcholine, nicotine and cytisine, and the antagonists alpha-bungarotoxin and neuronal bungarotoxin. The sensitivity of the nicotinic acetylcholine receptor for agonists followed the agonist potency rank-order: nicotine approximately acetylcholine > cytisine. The EC50 values of acetylcholine and nicotine are 78 microM and 76 microM, respectively. Equal concentrations of acetylcholine and nicotine induce inward currents with approximately the same peak amplitude whereas cytisine induces much smaller inward currents. Acetylcholine-induced currents are unaffected by high concentrations of alpha-bungarotoxin. Conversely, at 10 and 90 nM neuronal bungarotoxin reduces the amplitude of the 1 mM acetylcholine-induced inward current to 47% and 11% of control values, respectively. Both the agonist potency rank-order and the differential sensitivity to snake toxins of nicotinic receptors in N1E-115 cells are consistent with the known pharmacological profile of alpha 4 beta 2 nicotinic receptors expressed in Xenopus oocytes and distinct from those of all other nicotinic acetylcholine receptors of known functional subunit compositions. All data indicate that the native nicotinic acetylcholine receptor in N1E-115 cells is an assembly of alpha 4 and beta 2 subunits, the putative major subtype of nicotinic acetylcholine receptor in the brain.

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

NASA Astrophysics Data System (ADS)

Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

2006-01-01

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions.

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

PubMed Central

Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

2008-01-01

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions. PMID:19081771

Carbonate scale deactivating the biocathode in a microbial fuel cell

NASA Astrophysics Data System (ADS)

Santini, M.; Marzorati, S.; Fest-Santini, S.; Trasatti, S.; Cristiani, P.

2017-07-01

The development and the following inactivation of a carbon-based biocathode in single chamber and membraneless MFCs was investigated in this work. The electrochemical behavior of the biocathode has been analyzed over time during the MFC life. X-Ray Micro-Computed Tomographies (microCTs) have been carried out at progressive stages, documenting the building over time of a layer of scale deposition becoming thicker and thicker up to the cathode inactivation. The technique provides cross-sectional (tomographic) grayscale images and 3D reconstruction of volumes. Lighter color indicates lower X-ray attenuation (i.e., lower atomic density) thus allowing distinguishing biofilm from inorganic fouling on the basis of the average atomic number Z of each voxel (3D pixel). MicroCT was combined with Scanning Electron Microscopy (SEM) and Energy-Dispersive X-Ray Spectroscopy (EDX) in order to qualitatively recognize chemical species in each different layer of the cathode's section. Results correlated the presence of biofilm and calcium carbonate deposits, prevalently in the inner part of the cathode, with the produced electric current over time. A specific microCT-related software quantified the time-dependent carbonate scale deposition, identifying a correlation between the decreasing performances of the device and the increasing quantity of scale deposition that penetrates the cathode cross section in time.

Periodontal ligament versus bone marrow mesenchymal stem cells in combination with Bio-Oss scaffolds for ectopic and in situ bone formation: A comparative study in the rat.

PubMed

Yu, Bo-Han; Zhou, Qian; Wang, Zuo-Lin

2014-08-01

The aim of this study was to compare the osteogenic effects of periodontal ligament stem cells (PDLSCs) versus bone marrow mesenchymal stem cells (BMMSCs) in combination with Bio-Oss scaffolds on subcutaneous and critical-size defects in the immunodeficient rat calvarium. PDLSCs and BMMSCs were obtained from the same canine donor. Twenty-four rats were randomly assigned to one of four experimental groups (n = 6 each): group A (no-graft negative control), group B (Bio-Oss positive control), group C (BMMSC/Bio-Oss test group), and group D (PDLSC/Bio-Oss test group). Eight weeks post-transplantation, ectopic and in situ bone regeneration was evaluated by micro-computed tomography (µ-CT), histology, histomorphometry, and immunohistochemistry. The stem cell/Bio-Oss constructs were significantly superior to the controls in terms of their ability to promote osteogenesis (p < 0.01), while the PDLSC/Bio-Oss construct tended to be superior to the BMMSC/Bio-Oss construct. Thus, engineered stem cell/Bio-Oss complexes can successfully reconstruct critical-size defects in rats, and PDLSCs and BMMSCs are both suitable as seed cells. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Environmental Technology Verification Report: Climate Energy freewatt™ Micro-Combined Heat and Power System

EPA Science Inventory

The EPA GHG Center collaborated with the New York State Energy Research and Development Authority (NYSERDA) to evaluate the performance of the Climate Energy freewatt Micro-Combined Heat and Power System. The system is a reciprocating internal combustion (IC) engine distributed e...

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

PubMed

Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

2015-02-01

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

Micro-Nanomaterials for Tumor Microwave Hyperthermia: Design, Preparation, and Application.

PubMed

Chen, Xue; Tan, Longfei; Liu, Tianlong; Meng, Xianwei

2017-01-01

Cancer hyperthermia is attracting much attention in basic science and clinics. Among the hyperthermia techniques, microwave (MW) heating is most commonly used for cancer treatment. It offers highly competitive advantages: faster heat generation from microwave radiation, less susceptibility to heat up local tissues, maneuverability, and depth of penetration in tissues and capability of killing tumor cells. Although the encouraging clinical results are being collected, MW hyperthermia has its own challenges, such as inaccurate targeting and low selectivity, which lead to damage to the surrounding vital organs and tissues. To address these issues, this review aims to introduce micronanomaterials as promising agents for receiving the electromagnetic wave, which should be beneficial for improving the efficacy of MW hyperthermia. We have searched many peer-reviewed papers in medical and chemical material databases about micro-nanomaterials for tumor microwave hyperthermia. Distinguishing features and important progresses are introduced in this review. One hundred and forty papers were chosen and included in this review. Four parts were described, including hyperthermia techniques and the application of micro-nanomaterials, microwave thermal therapy and treatment principle, microwave absorbing micro-nanomaterials, the preparation and application of micro-nanomaterials in microwave thermal therapy. We review the most recent literatures on micro-nanomaterials-based MW heating strategies for cancer treatment, with the aim to give the reader an overview of the state-of-the-art of MW hyperthermia therapy. The future of MW responsive materials will also be discussed, including combination of imaging probes and targeting moieties. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Ionotropic and metabotropic glutamate receptor mediation of glucocorticoid-induced apoptosis in hippocampal cells and the neuroprotective role of synaptic N-methyl-D-aspartate receptors.

PubMed

Lu, J; Goula, D; Sousa, N; Almeida, O F X

2003-01-01

Glutamate receptors have been proposed to mediate the apoptotic actions of glucocorticoids in hippocampal cells. To further analyze the role of glutamate receptors in this process, we pretreated primary hippocampal cells from neonatal (postnatal day 4) rats with antagonists of ionotropic glutamate receptor (iGluR) and metabotropic glutamate receptor (mGluR) antagonists before exposure to the specific glucocorticoid receptor agonist dexamethasone (DEX) at a dose of 1 microM. Dizocilpine (MK801; a general N-methyl-D-aspartic acid [NMDA] receptor antagonist, NMDAR antagonist) and ifenprodil (a specific ligand of the NMDAR 2B subunit, NR2B), were used to block iGluR; (RS)-alpha-ethyl-4-carboxyphenylglycine (E4CPG) and (RS)-alpha-cyclopropyl-4-phosphonophenyl-glycine (CPPG) were employed as I/II (E4CPG) and II/III (CPPG) mGluR antagonists. Blockade of iGluR resulted in a significant attenuation of DEX-induced cell death; the finding that ifenprodil exerted a similar potency to MK801 demonstrates the involvement of NR2B receptors in glucocorticoid-induced cell death. Apoptosis accounted for a significant amount of the cell loss observed, as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling histochemistry for the in situ labeling of DNA breaks; apoptotic cells were distinguished from necrosis on the basis of morphological criteria, including chromatin condensation, membrane blebbing and presence of apoptotic bodies. Treatment with E4CPG and CPPG completely abolished the apoptotic response to DEX, thus showing the additional contribution of mGluR to the phenomenon. Further, dose-response studies with NMDA revealed that whereas high (10 microM) doses of NMDA themselves elicit cytotoxic responses, low (1-5 microM) concentrations of NMDA can effectively oppose DEX-induced cell death. Interestingly, the neuroprotective actions of low dose NMDA stimulation were abolished when either synaptic or extrasynaptic NMDA receptors were blocked with MK801 in combination with the GABA receptor antagonist bicuculline (synaptic) or ifenprodil (extrasynaptic). In summary, the present data show that both iGluR and mGluR mediate the neurotoxic effects of glucocorticoids on hippocampal cells and that pre-treatment with low doses of NMDA, by acting on synaptic and extrasynaptic receptors, render hippocampal cells less vulnerable to glucocorticoid insults.

A thermally self-sustained micro-power plant with integrated micro-solid oxide fuel cells, micro-reformer and functional micro-fluidic carrier

NASA Astrophysics Data System (ADS)

Scherrer, Barbara; Evans, Anna; Santis-Alvarez, Alejandro J.; Jiang, Bo; Martynczuk, Julia; Galinski, Henning; Nabavi, Majid; Prestat, Michel; Tölke, René; Bieberle-Hütter, Anja; Poulikakos, Dimos; Muralt, Paul; Niedermann, Philippe; Dommann, Alex; Maeder, Thomas; Heeb, Peter; Straessle, Valentin; Muller, Claude; Gauckler, Ludwig J.

2014-07-01

Low temperature micro-solid oxide fuel cell (micro-SOFC) systems are an attractive alternative power source for small-size portable electronic devices due to their high energy efficiency and density. Here, we report on a thermally self-sustainable reformer-micro-SOFC assembly. The device consists of a micro-reformer bonded to a silicon chip containing 30 micro-SOFC membranes and a functional glass carrier with gas channels and screen-printed heaters for start-up. Thermal independence of the device from the externally powered heater is achieved by exothermic reforming reactions above 470 °C. The reforming reaction and the fuel gas flow rate of the n-butane/air gas mixture controls the operation temperature and gas composition on the micro-SOFC membrane. In the temperature range between 505 °C and 570 °C, the gas composition after the micro-reformer consists of 12 vol.% to 28 vol.% H2. An open-circuit voltage of 1.0 V and maximum power density of 47 mW cm-2 at 565 °C is achieved with the on-chip produced hydrogen at the micro-SOFC membranes.

Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.

PubMed

Connell, Jodi L; Kim, Jiyeon; Shear, Jason B; Bard, Allen J; Whiteley, Marvin

2014-12-23

Microbes frequently live in nature as small, densely packed aggregates containing ∼10(1)-10(5) cells. These aggregates not only display distinct phenotypes, including resistance to antibiotics, but also, serve as building blocks for larger biofilm communities. Aggregates within these larger communities display nonrandom spatial organization, and recent evidence indicates that this spatial organization is critical for fitness. Studying single aggregates as well as spatially organized aggregates remains challenging because of the technical difficulties associated with manipulating small populations. Micro-3D printing is a lithographic technique capable of creating aggregates in situ by printing protein-based walls around individual cells or small populations. This 3D-printing strategy can organize bacteria in complex arrangements to investigate how spatial and environmental parameters influence social behaviors. Here, we combined micro-3D printing and scanning electrochemical microscopy (SECM) to probe quorum sensing (QS)-mediated communication in the bacterium Pseudomonas aeruginosa. Our results reveal that QS-dependent behaviors are observed within aggregates as small as 500 cells; however, aggregates larger than 2,000 bacteria are required to stimulate QS in neighboring aggregates positioned 8 μm away. These studies provide a powerful system to analyze the impact of spatial organization and aggregate size on microbial behaviors.

Solid oxide fuel cell short stack performance testing - Part A: Experimental analysis and μ-combined heat and power unit comparison

NASA Astrophysics Data System (ADS)

Mastropasqua, L.; Campanari, S.; Brouwer, J.

2017-12-01

The need to experimentally understand the detailed performance of SOFC stacks under operating conditions typical of commercial SOFC systems has prompted this two-part study. The steady state performance of a 6-cell short stack of yttria (Y2O3) stabilised zirconia (YSZ) with Ni/YSZ anodes and composite Sr-doped lanthanum manganite (LaMnO3, LSM)/YSZ cathodes is experimentally evaluated. In Part A, the stack characterisation is carried out by means of sensitivity analyses on the fuel utilisation factor and the steam-to-carbon ratio. Electrical and environmental performances are assessed and the results are compared with a commercial full-scale micro-CHP system, which comprises the same cells. The results show that the measured temperature dynamics of the short stack in a test stand environment are on the order of many minutes; therefore, one cannot neglect temperature dynamics for a precise measurement of the steady state polarisation behaviour. The overall polarisation performance is comparable to that of the full stack employed in the micro-CHP system, confirming the good representation that short-stack analyses can give of the entire SOFC module. The environmental performance is measured verifying the negligible values of NO emissions (<10 ppb) across the whole polarisation curve.

The Role of Nutraceuticals in Pancreatic Cancer Prevention and Therapy: Targeting Cellular Signaling, MicroRNAs, and Epigenome.

PubMed

Li, Yiwei; Go, Vay Liang W; Sarkar, Fazlul H

2015-01-01

Pancreatic cancer is one of the most aggressive malignancies in US adults. Experimental studies have found that antioxidant nutrients could reduce oxidative DNA damage, suggesting that these antioxidants may protect against pancreatic carcinogenesis. Several epidemiologic studies showed that dietary intake of antioxidants was inversely associated with the risk for pancreatic cancer, demonstrating the inhibitory effects of antioxidants on pancreatic carcinogenesis. Moreover, nutraceuticals, the anticancer agents from diet or natural plants, have been found to inhibit the development and progression of pancreatic cancer through the regulation of cellular signaling pathways. Importantly, nutraceuticals also up-regulate the expression of tumor-suppressive microRNAs (miRNAs) and down-regulate the expression of oncogenic miRNAs, leading to the inhibition of pancreatic cancer cell growth and pancreatic cancer stem cell self-renewal through modulation of cellular signaling network. Furthermore, nutraceuticals also regulate epigenetically deregulated DNAs and miRNAs, leading to the normalization of altered cellular signaling in pancreatic cancer cells. Therefore, nutraceuticals could have much broader use in the prevention and/or treatment of pancreatic cancer in combination with conventional chemotherapeutics. However, more in vitro mechanistic experiments, in vivo animal studies, and clinical trials are needed to realize the true value of nutraceuticals in the prevention and/or treatment of pancreatic cancer.

The pathogenic role of persistent milk signaling in mTORC1- and milk-microRNA-driven type 2 diabetes mellitus.

PubMed

Melnik, Bodo C

2015-01-01

Milk, the secretory product of the lactation genome, promotes growth of the newborn mammal. Milk delivers insulinotropic amino acids, thus maintains a molecular crosstalk with the pancreatic β-cell of the milk recipient. Homeostasis of β-cells and insulin production depend on the appropriate magnitude of mTORC1 signaling. mTORC1 is activated by branched-chain amino acids (BCAAs), glutamine, and palmitic acid, abundant nutrient signals of cow´s milk. Furthermore, milk delivers bioactive exosomal microRNAs. After milk consumption, bovine microRNA-29b, a member of the diabetogenic microRNA-29- family, reaches the systemic circulation and the cells of the milk consumer. MicroRNA-29b downregulates branchedchain α-ketoacid dehydrogenase, a potential explanation for increased BCAA serum levels, the metabolic signature of insulin resistance and type 2 diabetes mellitus (T2DM). In non-obese diabetic mice, microRNA-29b downregulates the antiapoptotic protein Mcl-1, which leads to early β-cell death. In all mammals except Neolithic humans, milk-driven mTORC1 signaling is physiologically restricted to the postnatal period. In contrast, chronic hyperactivated mTORC1 signaling has been associated with the development of age-related diseases of civilization including T2DM. Notably, chronic hyperactivation of mTORC1 enhances endoplasmic reticulum stress that promotes apoptosis. In fact, hyperactivated β-cell mTORC1 signaling induced early β-cell apoptosis in a mouse model. The EPIC-InterAct Study demonstrated an association between milk consumption and T2DM in France, Italy, United Kingdom, Germany, and Sweden. In contrast, fermented milk products and cheese exhibit an inverse correlation. Since the early 1950´s, refrigeration technology allowed widespread consumption of fresh pasteurized milk, which facilitates daily intake of bioactive bovine microRNAs. Persistent uptake of cow´s milk-derived microRNAs apparently transfers an overlooked epigenetic diabetogenic program that should not reach the human food chain.

Accomplished Teaching: Using Video Recorded Micro-Teaching Discourse to Build Candidate Teaching Competencies

ERIC Educational Resources Information Center

Shaw, Denise

2017-01-01

Objectives of this articles are to present the findings of video recorded communication between teacher candidates and peers during simulated micro-teaching. The micro-teaching activity in its entirety combines conventional face-to-face interaction, video micro-teaching, peer and instructor feedback, alongside self-reflection to undergird the…

Combining micro-structures and micro-algae to increase lipid production for bio-fuel

NASA Astrophysics Data System (ADS)

Vyawahare, Saurabh; Zhu, Emilly; Mestler, Troy; Estévez-Torres, André.; Austin, Robert

2011-03-01

3rd generation bio-fuels like lipid producing micro-algae are a promising source of energy that could replace our dependence on petroleum. However, until there are improvements in algae oil yields, and a reduction in the energy needed for processing, algae bio-fuels are not economically competitive with petroleum. Here, we describe our work combining micro-fabricated devices with micro-algae Neochloris oleoabundans, a species first isolated on the sand dunes of Saudi Arabia. Inserting micro-algae of varying fitness into a landscape of micro-habitats allows us to evolve and select them based on a variety of conditions like specific gravity, starvation response and Nile Red fluorescence (which is a marker for lipid production). Hence, we can both estimate the production of lipids and generate conditions that allow the creation and isolation of algae which produce higher amounts of lipids, while discarding the rest. Finally, we can use micro-fabricated structures and flocculation to de-water these high lipid producing algae, reducing the need for expensive centrifugation and filtration.

Renal cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes in primary cultures of human and rat proximal tubular cells.

PubMed

McGoldrick, Trevor A; Lock, Edward A; Rodilla, Vicente; Hawksworth, Gabrielle M

2003-07-01

Proximal tubular cells from human (HPT) and rat (RPT) kidneys were isolated, grown to confluence and incubated with S-(1,2-dichlorovinyl)- l-cysteine (DCVC), S-(1,2,2-trichlorovinyl)- l-cysteine (TCVC), S-(1,1,2,2-tetrafluoroethyl)- l-cysteine (TFEC) and S-(2-chloro-1,1-difluorethyl)- l-cysteine (CDFEC), the cysteine conjugates of nephrotoxicants. The cultures were exposed to the conjugates for 12, 24 and 48 h and the toxicity determined using the MTT assay. All four conjugates caused dose-dependent toxicity to RPT cells over the range 50-1,000 microM, the order of toxicity being DCVC>TCVC>TFEC=CDFEC. The inclusion of aminooxyacetic acid (AOAA; 250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as C-S lyase, afforded protection, indicating that C-S lyase has a role in the bioactivation of these conjugates. In HPT cultures only DCVC caused significant time- and dose-dependent toxicity. Exposure to DCVC (500 microM) for 48 h decreased cell viability to 7% of control cell values, whereas co-incubation of DCVC (500 microM) with AOAA (250 microM) resulted in cell viability of 71%. Human cultures were also exposed to S-(1,2-dichlorovinyl)-glutathione (DCVG). DCVG was toxic to HPT cells, but the onset of toxicity was delayed compared with the corresponding cysteine conjugate. AOAA afforded almost complete protection from DCVG toxicity. Acivicin (250 microM), an inhibitor of gamma-glutamyl transferase (gamma-GT), partially protected against DCVG (500 microM)-induced toxicity at 48 h (5% viability and 53% viability in the absence and presence of acivicin, respectively). These results suggest that DCVG requires processing by gamma-GT prior to bioactivation by C-S lyase in HPT cells. The activity of C-S lyase, using TFEC as a substrate, and glutamine transaminase K (GTK) was measured in rat and human cells with time in culture. C-S lyase activity in RPT and HPT cells decreased to approximately 30% of fresh cell values by the time the cells reached confluence (120 h), whereas the decline in GTK activity was less marked (50% of the fresh cell values at confluence). Rat cells had threefold higher activity than human cells at each time point. This higher activity may partly explain the differences in toxicity between rat and human proximal tubular cells in culture.

Glucocorticoid-Mediated Repression of the Oncogenic microRNA Cluster miR-17∼92 Contributes to the Induction of Bim and Initiation of Apoptosis

PubMed Central

Molitoris, Jason K.; McColl, Karen S.

2011-01-01

Synthetic glucocorticoids were one of the first effective treatments for lymphoid malignancies because of their ability to induce apoptosis and are still used in combination with other chemotherapeutic agents. Up-regulation of Bim, a proapoptotic member of the B-cell lymphoma-2 family, is an important mediator of glucocorticoid-induced apoptosis. Although glucocorticoids are known to elevate Bim mRNA and protein, little is known about the mechanism. Here, we report that glucocorticoids repress the expression of the microRNA cluster miR-17∼92, which results in elevated Bim protein expression as a mechanism by which glucocorticoids induce Bim. Using a luciferase-Bim 3′ untranslated region construct, we demonstrate that glucocorticoids mediate Bim induction posttranscriptionally after miR-17∼92 repression, resulting in increased Bim protein expression. Overexpression of miR-17∼92 microRNAs decreases Bim induction and attenuates glucocorticoid-mediated apoptosis. Conversely, knockdown of miR-17∼92 increases Bim protein expression and glucocorticoid-mediated apoptosis. These findings indicate that endogenous levels of miR-17∼92 repress Bim expression in T-cell lymphoid malignancies and that glucocorticoids induce Bim expression via down-regulation of the miR-17∼92 microRNA cluster. Our findings present a novel mechanism that contributes to the up-regulation of Bim and induction of apoptosis in lymphocytes after glucocorticoid treatment. Furthermore, our work demonstrating that inhibition of miR-17∼92 increases glucocorticoid-induced apoptosis highlights the potential importance of miR-17∼92 as a therapeutic target in leukemias and lymphomas. PMID:21239610

Multisensor Instrument for Real-Time Biological Monitoring

NASA Technical Reports Server (NTRS)

Zhang, Sean (Zhanxiang); Xu, Guoda; Qiu, Wei; Lin, Freddie

2004-01-01

The figure schematically depicts an instrumentation system, called a fiber optic-based integration system (FOBIS), that is undergoing development to enable real-time monitoring of fluid cell cultures, bioprocess flows, and the like. The FOBIS design combines a micro flow cytometer (MFC), a microphotometer (MP), and a fluorescence-spectrum- or binding-force-measuring micro-sensor (MS) in a single instrument that is capable of measuring multiple biological parameters simultaneously or sequentially. The fiber-optic-based integration system is so named because the MFC, the MP, and the MS are integrated into a single optical system that is coupled to light sources and photometric equipment via optical fibers. The optical coupling components also include a wavelength-division multiplexer and diffractive optical elements. The FOBIS includes a laserdiode- and fiber-optic-based optical trapping subsystem (optical tweezers ) with microphotometric and micro-sensing capabilities for noninvasive confinement and optical measurement of relevant parameters of a single cell or other particle. Some of the measurement techniques implemented together by the FOBIS have long been used separately to obtain basic understanding of the optical properties of individual cells and other organisms, the optical properties of populations of organisms, and the interrelationships among these properties, physiology of the organisms, and physical processes that govern the media that surround the organisms. For example, flow cytometry yields information on numerical concentrations, cross-sectional areas, and types of cells or other particles. Micro-sensing can be used to measure pH and concentrations of oxygen, carbon dioxide, glucose, metabolites, calcium, and antigens in a cell-culture fluid, thereby providing feedback that can be helpful in improving control over a bioprocess. Microphotometry (including measurements of scattering and fluorescence) can yield further information about optically trapped individual particles. In addition to the multifunctionality not previously available in a single biological monitoring system, the FOBIS offers advantages of low mass, sensitivity, accuracy, portability, low cost, compactness (the overall dimensions of the fully developed FOBIS sensor head are expected to be less than 1 by 1 by 2 cm), and immunity to electromagnetic interference at suboptical frequencies. FOBIS could be useful in a variety of laboratory and field settings in such diverse endeavors as medical, veterinary, and general biological research; medical and veterinary diagnosis monitoring of industrial bioprocesses; and analysis of biological contaminants in air, water, and food.

Sulforaphane induces apoptosis in rhabdomyosarcoma and restores TRAIL-sensitivity in the aggressive alveolar subtype leading to tumor elimination in mice

PubMed Central

Bergantin, Elisa; Quarta, Carmelo; Nanni, Cristina; Fanti, Stefano; Pession, Andrea; Cantelli-Forti, Giorgio; Tonelli, Roberto; Hrelia, Patrizia

2014-01-01

Rhadbomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is subdivided in the embryonal (ERMS) and alveolar (ARMS) subtypes, the latter being associated with the worst prognosis. We report that sulforaphane (SFN), a broccoli-derived anticancer isothiocyanate, causes dose- and time-dependent growth inhibition and apoptosis in both ERMS and ARMS cells. In ARMS, SFN induced the modulation of expression of crucial genes and proteins: mRNA and protein levels of PAX3-FKHR, MYCN, and MET decreased, while those of p21 and TRAIL-receptor DR5 (but not DR4) increased. Since DR5 expression increased specifically in ARMS, we treated ARMS cells with TRAIL, SFN, or their combination. While ARMS cells (RH30 and RH4) proved to be TRAIL-resistant, SFN restored their sensitivity to TRAIL-induced cell-growth inhibition, leading to a stronger effect in combination with TRAIL. ARMS cells transfected with siDR5 showed that SFN-induced DR5 acts as a key regulator, being directly related to the TRAIL-induced cell-growth inhibition. The in vivo anti-tumor activity of SFN and TRAIL was evaluated in a xenograft murine model of ARMS through microPET. The results showed that the systemic treatment (3 wk) of mice with SFN or TRAIL as single agents only delayed tumor evolution, while the combined treatment of SFN and TRAIL led to tumor elimination. These findings indicate that SFN triggers the apoptotic pathway in both alveolar and embryonal rhabdomyosarcomas and that combined treatment with SFN and TRAIL might be a promising therapy for the aggressive alveolar subtype. PMID:24971463

Viral microRNA effects on persistent infection of human lymphoid cells by polyomavirus SV40

PubMed Central

McNees, Adrienne L.; Harrigal, Lindsay J.; Kelly, Aoife; Minard, Charles G.; Wong, Connie

2018-01-01

Background Polyomaviruses, including simian virus 40 (SV40), display evidence of lymphotropic properties. This study analyzed the nature of SV40–human lymphocyte interactions in established cell lines and in primary lymphocytes. The effects of viral microRNA and the structure of the viral regulatory region on SV40 persistence were examined. Results SV40 DNA was maintained in infected B cell and myeloid cell lines during cell growth for at least 28 days. Limiting dilution analysis showed that low amounts of SV40 DNA (~2 copies per cell) were retained over time. Infected B cells remained viable and able to proliferate. Genome copies of the SV40 microRNA-null mutant persisted at higher levels than the DNA of wild-type viruses. Complex viral regulatory regions produced modestly higher DNA levels than simple regulatory regions. Viral large T-antigen protein was detected at low frequency and at low levels in infected B cells. Following infection of primary lymphocytes, SV40 DNA was detected in CD19+ B cells and CD14+ monocytes, but not in CD3+ T cells. Rescue attempts using either lysates of SV40-infected B lymphocytes, coculture of live cells, or infectious center assays all showed that replication-competent SV40 could be recovered on rare occasions. SV40 infections altered the expression of several B cell surface markers, with more pronounced changes following infections with the microRNA-null mutant. Conclusion These findings indicate that SV40 can establish persistent infections in human B lymphocytes. The cells retain low copy numbers of viral DNA; the infections are nonproductive and noncytolytic but can occasionally produce infectious virus. SV40 microRNA negatively regulates the degree of viral effects on B cells. Significance Lymphocytes may serve as viral reservoirs and may function to disseminate polyomaviruses to different tissues in a host. To our knowledge, this report is the first extensive analysis of viral microRNA effects on SV40 infection of human lymphocytes. PMID:29432481

A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate

PubMed Central

Lozano-Velasco, Estefanía; Vallejo, Daniel; Esteban, Francisco J.; Doherty, Chris; Hernández-Torres, Francisco; Franco, Diego

2015-01-01

The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5+ satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine. PMID:26055324

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

PubMed

Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

2001-12-13

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.

Biological and Mechanical Effects of Micro-Nanostructured Titanium Surface on an Osteoblastic Cell Line In vitro and Osteointegration In vivo.

PubMed

Hao, Jingzu; Li, Ying; Li, Baoe; Wang, Xiaolin; Li, Haipeng; Liu, Shimin; Liang, Chunyong; Wang, Hongshui

2017-09-01

Hybrid micro-nanostructure implant surface was produced on titanium (Ti) surface by acid etching and anodic oxidation to improve the biological and mechanical properties. The biological properties of the micro-nanostructure were investigated by simulated body fluid (SBF) soaking test and MC3T3-E1 cell co-culture experiment. The cell proliferation, spreading, and bone sialoprotein (BSP) gene expression were examined by MTT, SEM, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the mechanical properties were evaluated by instrumented nanoindentation test and friction-wear test. Furthermore, the effect of the micro-nanostructure surface on implant osteointegration was examined by in vivo experiment. The results showed that the formation of bone-like apatite was accelerated on the micro-nanostructured Ti surface after immersion in simulated body fluid, and the proliferation, spreading, and BSP gene expression of the MC3T3-E1 cells were also upregulated on the modified surface. The micro-nanostructured Ti surface displayed decreased friction coefficient, stiffness value, and Young's modulus which were much closer to those of the cortical bone, compared to the polished Ti surface. This suggested much better mechanical match to the surrounding bone tissue of the micro-nanostructured Ti surface. Furthermore, the in vivo animal experiment showed that after implantation in the rat femora, the micro-nanostructure surface displayed higher bonding strength between bone tissues and implant; hematoxylin and eosin (H&E) staining suggested that much compact osteoid tissue was observed at the interface of Micro-nano-Ti-bone than polished Ti-bone interface after implantation. Based on these results mentioned above, it was concluded that the improved biological and mechanical properties of the micro-nanostructure endowed Ti surface with good biocompatibility and better osteointegration, implying the enlarged application of the micro-nanostructure surface Ti implants in future.

Development of a Dynamic Operational Scheduling Algorithm for an Independent Micro-Grid with Renewable Energy

NASA Astrophysics Data System (ADS)

Obara, Shin'ya

A micro-grid with the capacity for sustainable energy is expected to be a distributed energy system that exhibits quite a small environmental impact. In an independent micro-grid, “green energy,” which is typically thought of as unstable, can be utilized effectively by introducing a battery. In the past study, the production-of-electricity prediction algorithm (PAS) of the solar cell was developed. In PAS, a layered neural network is made to learn based on past weather data and the operation plan of the compound system of a solar cell and other energy systems was examined using this prediction algorithm. In this paper, a dynamic operational scheduling algorithm is developed using a neural network (PAS) and a genetic algorithm (GA) to provide predictions for solar cell power output. We also do a case study analysis in which we use this algorithm to plan the operation of a system that connects nine houses in Sapporo to a micro-grid composed of power equipment and a polycrystalline silicon solar cell. In this work, the relationship between the accuracy of output prediction of the solar cell and the operation plan of the micro-grid was clarified. Moreover, we found that operating the micro-grid according to the plan derived with PAS was far superior, in terms of equipment hours of operation, to that using past average weather data.

The use of microRNA by human viruses: lessons from NK cells and HCMV infection.

PubMed

Goldberger, Tal; Mandelboim, Ofer

2014-11-01

Depending on ethnicity and on social conditions, between 40 and 90 % of the population is infected with human cytomegalovirus (HCMV). In immunocompetent patients, the virus may cause an acute disease and then revert to a state of latency, which enables its coexistence with the human host. However, in cases of immunosuppression or in neonatal infections, HCMV can cause serious long-lasting illnesses. HCMV has developed multiple mechanisms in order to escape its elimination by the immune system, specifically by two killer cell types of the adaptive and the innate immune systems; cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, respectively. Another fascinating aspect of HCMV is that like other highly developed herpesviruses, it expresses its own unique set of microRNAs. Here, we initially describe how the activity of NK cells is regulated under normal conditions and during infection. Then, we discuss what is currently known about HCMV microRNA-mediated interactions, with special emphasis on immune modulation and NK cell evasion. We further illustrate the significant modulation of cellular microRNAs during HCMV infection. Although, the full target spectrum of HCMV microRNAs is far from being completely elucidated, it can already be concluded that HCMV uses its "multitasking" microRNAs to globally affect its own life cycle, as well as important cellular and immune-related pathways.

Micro-Ring Structures Stabilize Microdroplets to Enable Long Term Spheroid Culture in 384 Hanging Drop Array Plates

PubMed Central

Hsiao, Amy Y.; Tung, Yi-Chung; Kuo, Chuan-Hsien; Mosadegh, Bobak; Bedenis, Rachel; Pienta, Kenneth J.; Takayama, Shuichi

2012-01-01

Using stereolithography, 20 different structural variations comprised of millimeter diameter holes surrounded by trenches, plateaus, or micro-ring structures were prepared and tested for their ability to stably hold arrays of microliter sized droplets within the structures over an extended period of time. The micro-ring structures were the most effective in stabilizing droplets against mechanical and chemical perturbations. After confirming the importance of micro-ring structures using rapid prototyping, we developed an injection molding tool for mass production of polystyrene 3D cell culture plates with an array of 384 such micro-ring surrounded through-hole structures. These newly designed and injection molded polystyrene 384 hanging drop array plates with micro-rings were stable and robust against mechanical perturbations as well as surface fouling-facilitated droplet spreading making them capable of long term cell spheroid culture of up to 22 days within the droplet array. This is a significant improvement over previously reported 384 hanging drop array plates which are susceptible to small mechanical shocks and could not reliably maintain hanging drops for longer than a few days. With enhanced droplet stability, the hanging drop array plates with micro-ring structures provide better platforms and open up new opportunities for high-throughput preparation of microscale 3D cell constructs for drug screening and cell analysis. PMID:22057945

Micro-ring structures stabilize microdroplets to enable long term spheroid culture in 384 hanging drop array plates.

PubMed

Hsiao, Amy Y; Tung, Yi-Chung; Kuo, Chuan-Hsien; Mosadegh, Bobak; Bedenis, Rachel; Pienta, Kenneth J; Takayama, Shuichi

2012-04-01

Using stereolithography, 20 different structural variations comprised of millimeter diameter holes surrounded by trenches, plateaus, or micro-ring structures were prepared and tested for their ability to stably hold arrays of microliter sized droplets within the structures over an extended period of time. The micro-ring structures were the most effective in stabilizing droplets against mechanical and chemical perturbations. After confirming the importance of micro-ring structures using rapid prototyping, we developed an injection molding tool for mass production of polystyrene 3D cell culture plates with an array of 384 such micro-ring surrounded through-hole structures. These newly designed and injection molded polystyrene 384 hanging drop array plates with micro-rings were stable and robust against mechanical perturbations as well as surface fouling-facilitated droplet spreading making them capable of long term cell spheroid culture of up to 22 days within the droplet array. This is a significant improvement over previously reported 384 hanging drop array plates which are susceptible to small mechanical shocks and could not reliably maintain hanging drops for longer than a few days. With enhanced droplet stability, the hanging drop array plates with micro-ring structures provide better platforms and open up new opportunities for high-throughput preparation of microscale 3D cell constructs for drug screening and cell analysis.

Parallel multipoint recording of aligned and cultured neurons on corresponding Micro Channel Array toward on-chip cell analysis.

PubMed

Tonomura, W; Moriguchi, H; Jimbo, Y; Konishi, S

2008-01-01

This paper describes an advanced Micro Channel Array (MCA) so as to record neuronal network at multiple points simultaneously. Developed MCA is designed for neuronal network analysis which has been studied by co-authors using MEA (Micro Electrode Arrays) system. The MCA employs the principle of the extracellular recording. Presented MCA has the following advantages. First of all, the electrodes integrated around individual micro channels are electrically isolated for parallel multipoint recording. Sucking and clamping of cells through micro channels is expected to improve the cellular selectivity and S/N ratio. In this study, hippocampal neurons were cultured on the developed MCA. As a result, the spontaneous and evoked spike potential could be recorded by sucking and clamping the cells at multiple points. Herein, we describe the successful experimental results together with the design and fabrication of the advanced MCA toward on-chip analysis of neuronal network.

Tracking microbial colonization patterns associated with micro-environments of rice

NASA Astrophysics Data System (ADS)

Schmidt, Hannes; Eickhorst, Thilo

2015-04-01

The interface between soil and roots (i.e. the rhizosphere) represents a highly dynamic micro-environment for microbial populations. Root-derived compounds are released into the rhizosphere and may attract, stimulate, or inhibit native soil microorganisms. Microbes associated with the rhizosphere, in turn, may have deleterious, neutral, or promoting effects on the plant. Such influences of microbial populations on the plant and vice versa are likely to be greatest in close vicinity to the root surface. It is therefore essential to detect and visualize preferential micro-sites of microbial root colonization to identify potential areas of microbe-plant interaction. We present a single-cell based approach allowing for the localization, quantification, and visualization of native microbial populations in the rhizosphere and on the rhizoplane of soil-grown roots in situ. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with confocal laser scanning microscopy was applied to observe colonization densities and patterns of microbial populations associated with wetland rice. Hybridizations with domain- and phylum-specific oligonucleotide probes showed that the growth stage of the rice plant as well as the distance to the root surface had a strong influence on microbial colonization patterns. Three-dimensional visualizations of root-associated microbes revealed micro-sites of preferential colonization. Highest cell numbers of archaea and bacteria were found at flowering stage of rice plant development. Irregular distribution patterns of microbiota observed at early growth stages shifted towards more uniform colonization with plant age. Accordingly, the highest colonization densities shifted from the tip to more mature regions of rice roots. Methanogenic archaea and methanotrophic bacteria were found to be co-localized at basal regions of lateral roots. Beneficial effects of a close association with root surfaces were indicated by proportionally higher numbers of methane-oxidizing bacteria on the rhizoplane compared to the rhizosphere. Such spatial effects could not be observed for methanogenic archaea. As a consequence, the detection and visualization of microbial colonization patterns on a micro-scale via CARD-FISH represents an instrumental approach in revealing potential sites of interaction between microbes and plants in soil micro-environments.

Hemolysis of human red blood cells induced by the combination of diethyldithiocarbamate (DDC) and divalent metals: modulation by anaerobiosis, certain antioxidants and oxidants.

PubMed

Ginsburg, I; Sadovnic, M; Varani, J; Tirosh, O; Kohen, R

1999-08-01

The objective of the present communication is to describe the role played by combinations between diethydithiocarbamate (DDC) and divalent metals in hemolysis of human RBC. RBC which had been treated with DDC (10-50 microM) were moderately hemolyzed (about 50%) upon the addition of subtoxic amounts of Cu2+ (50 microM). However, a much stronger and a faster hemolysis occurred either if mixtures of RBC-DDC were immediately treated either by Co2+ (50 microM) or by a premixture of Cu2+ and Co2+ (Cu:Co) (50 microM). While Fe2+ and Ni2+, at 50 microM, initiated 30-50% hemolysis when combined with DDC (50 microM), on a molar basis, Cd2+ was at least 50 fold more efficient than any of the other metals in the initiation of hemolysis by DDC. On the other hand, neither Mn2+ nor Zn2+, had any hemolysis-initiating effects. Co2+ was the only metal which totally blocked hemolysis if added to DDC prior to the addition of the other metals. Hemolysis by mixtures of DDC + (Cu:Co) was strongly inhibited by anaerobiosis (flushing with nitrogen gas), by the reducing agents glutathione, N-acetyl cysteine, mercaptosuccinate, ascorbate, TEMPO, and alpha-tocopherol, by the PLA2 inhibitorbromophenacylbromide (BrPACBr), by tetracycline as well as by phosphatidyl choline, cholesterol and by trypan blue. However, TEMPO, BrPACBr and PC were the only agents which inhibited hemolysis induced by DDC: Cd2+ complexes. On the other hand, none of the classical scavengers of reactive oxygen species (ROS) employed e.g dimethylthiourea, catalase, histidine, mannitol, sodium benzoate, nor the metal chelators desferal and phenanthroline, had any appreciable inhibitory effects on hemolysis induced by DDC + (Cu:Co). DDC oxidized by H2O2 lost its capacity to act in concert either with Cu2+ or with Cd2+ to hemolyze RBC. While either heating RBC to temperatures greater than 37 degrees C or exposure of the cells to glucose-oxidase-generated peroxide diminished their susceptibility to hemolysis, exposure to the peroxyl radical from AAPH, enhanced hemolysis by DDC + (Cu:Co). The cyclovoltammetry patterns of DDC were drastically changed either by Cu2+, Co2+ or by Cd2+ suggesting a strong interaction of the metals with DDC. Also, while the absorbance spectrum of DDC at 280 nm was decreased by 50% either by Co2+, Cd2+ or by H2O2, a 90% reduction in absorbance occurred if DDC + H2O2 mixtures were treated either by Cu2+ or by Co2+, but not by Cd2+. Taken together, it is suggested that DDC-metal chelates can induce hemolysis by affecting the stability and the integrity of the RBC membrane, and possibly also of the cytoskeleton and the role played by reducing agents as inhibitors might be related to their ability to deplete oxygen which is also supported by the inhibitory effects of anaeobiosis.

Absence of a universal mechanism of mitochondrial toxicity by nucleoside analogs.

PubMed

Lund, Kaleb C; Peterson, LaRae L; Wallace, Kendall B

2007-07-01

Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 microM; 2.7 and 13.4 microg/ml), didanosine (ddI) (10 and 50 microM; 2.4 and 11.8 microg/ml), and zalcitabine (ddC) (1 and 5 microM; 0.21 and 1.1 microg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 microg/ml; 1.3 microM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 microM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard.

Multi-Photon Micro-Spectroscopy of Biological Specimens

DTIC Science & Technology

2000-07-01

Micro-spectroscopy, multi-photon fluorescence spectroscopy, second harmonic generation, plant tissues, stem, chloroplast, protoplast, maize, Arabidopsis...harmonic generation (SHG) in the plant cell 5wall. In this case, micro-spectroscopy provides a means of verification that, indeed, SHG occurs in plant ...fluorescence microscopy -the response of plant cells to high intensity illumination," Micron (in press) 2000. 3. H.-C. Huang and C. -C Chen, "Genome

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes.

PubMed

Jin, Ya; Yuan, Qi; Zhang, Jun; Manabe, Takashi; Tan, Wen

2015-09-01

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an "overlap score," (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 "overlap factors," (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MicroRNA-320a suppresses human colon cancer cell proliferation by directly targeting {beta}-catenin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jian-Yong; State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 710032 Xi'an; Huang, Yi

2012-04-20

Highlights: Black-Right-Pointing-Pointer miR-320a is downregulated in human colorectal carcinoma. Black-Right-Pointing-Pointer Overexpression of miR-320a inhibits colon cancer cell proliferation. Black-Right-Pointing-Pointer {beta}-Catenin is a direct target of miR-320a in colon cancer cells. Black-Right-Pointing-Pointer miR-320a expression inversely correlates with mRNA expression of {beta}-catenin's target genes in human colon carcinoma. -- Abstract: Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-320a) in human colorectal carcinoma. However, its expression pattern and underlying mechanisms in the development and progression of colorectal carcinoma has not been elucidated clearly. Here, we performed real-time PCR to examine the expression levels of miR-320a in colonmore » cancer cell lines and tumor tissues. And then, we investigated its biological functions in colon cancer cells by a gain of functional strategy. Further more, by the combinational approaches of bioinformatics and experimental validation, we confirmed target associations of miR-320a in colorectal carcinoma. Our results showed that miR-320a was frequently downregulated in cancer cell lines and colon cancer tissues. And we demonstrated that miR-320a restoration inhibited colon cancer cell proliferation and {beta}-catenin, a functionally oncogenic molecule was a direct target gene of miR-320a. Finally, the data of real-time PCR showed the reciprocal relationship between miR-320a and {beta}-catenin's downstream genes in colon cancer tissues. These findings indicate that miR-320a suppresses the growth of colon cancer cells by directly targeting {beta}-catenin, suggesting its application in prognosis prediction and cancer treatment.« less