Identification of novel modulators of WNT/β-catenin signalling in articular chondrocytes by large scale siRNA screening

Chondrocytes in articular cartilage synthesize collagen type II and large sulfated proteoglycans, whereas the same cells cultured in monolayer (2D) dedifferentiate into fibroblastic cells and express collagen type I and small proteoglycans. On the other hand, a pellet culture system was developed as a method for preventing the phenotypic modulation of chondrocytes and promoting the redifferentiation of dedifferentiated ones. Because the pellet culture system forms only one cell aggregate each tube by a centrifugator, the pellet could not be applied to produce a tissue-engineered cartilage. Therefore, we tried to form chondrocyte aggregates by a rotational culture, expecting to form a large number of aggregates at once. In order to increase cell–cell interactions and decrease chondrocyte–material interaction, dishes with low retention of protein adsorption and cell adhesiveness were used. In addition, rotational shaking of the dish including cells was attempted to increase the cell–cell interaction. The shaking speed was set at 80 rpm, so the cells would be distributed in the center of the dish to augment the frequency of cell–cell contact. Under these conditions, bovine articular chondrocytes started aggregating in a few hours. At 24–36 h of rotational culture, aggregates with smooth surfaces were observed. Parameters such as increase of culture time and addition of TGF-β controlled diameters of the aggregates. There were many fusiform cells at the periphery of the aggregates, where the cells tended to form a multilayered zone in cross sections. In addition, lacune-like structure, which was almost the same as pellet culture, was observed. It was found that the internal structure of the aggregates was similar to that of pellets reported previously. Therefore, the aggregates formed by a rotational culture could become an essential component to make tissue-engineered artificial cartilage.

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Proper expression of helix–loop–helix protein Id2 is important to chondrogenic differentiation of ATDC5 cells

Biochemical Journal ◽

10.1042/bj20081715 ◽

2009 ◽

Vol 419 (3) ◽

pp. 635-643 ◽

Cited By ~ 3

Author(s):

Liu Yang ◽

Xiaoyun Ma ◽

Anne Lyone ◽

Junhui Zou ◽

Michael L. Blackburn ◽

...

Keyword(s):

Chondrogenic Differentiation ◽

Large Scale ◽

Articular Chondrocytes ◽

Type Ii Collagen ◽

Inverse Pcr ◽

Pcr Analysis ◽

Insertion Mutagenesis ◽

Chondroprogenitor Cells

The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. To identify novel factors that are involved in the control of chondrogenesis, we carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 (inhibitor of DNA-binding protein 2) gene. This retroviral insertion increased Id2 protein levels to twice those found in normal ATDC5 cells. To investigate whether an elevated level of Id2 protein was responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopic Id2 exhibited signs of de-differentiation, such as rapid growth, and insulin failed to induce expression of Sox9 (Sry-type high-mobility-group box 9) or matrix genes such as type II collagen (COL2) in these cells. When endogenous Id2 was knocked down by siRNA (small interfering RNA) in ATDC5 cells, expression of Sox9 and COL2 was increased and chondrogenic differentiation was accelerated. To examine how Id2 is expressed in chondrocytes in vivo, we carried out immunostaining of E16.5 mouse embryos and found that Id2 is expressed in articular chondrocytes and proliferating chondrocytes, but barely detectable in hypertrophic chondrocytes. Our results suggest that proper expression of Id2 is important to achieving a fine balance between growth and differentiation during chondrogenesis.

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High-throughput siRNA screening reveals functional interactions and redundancies of human processive exoribonucleases

10.1101/2020.08.05.238006 ◽

2020 ◽

Author(s):

Anna Hojka-Osinska ◽

Aleksander Chlebowski ◽

Ewelina P. Owczarek ◽

Kamila Afek ◽

Kamila Kłosowska ◽

...

Keyword(s):

Rna Splicing ◽

Large Scale ◽

Rna Metabolism ◽

Rna Degradation ◽

Rna Decay ◽

Positive Interactions ◽

Microtubule Organization ◽

Functional Relationships ◽

Functional Interactions ◽

Sirna Screening

ABSTRACTProcessive exoribonucleases, the executors of RNA decay, participate in multiple physical and functional interactions. Unlike physical ones, functional relationships have not been investigated in human cells. Here we have screened cells deficient in DIS3, XRN2, EXOSC10, DIS3L, and DIS3L2 with a custom siRNA library and determined their functional interactions with diverse pathways of RNA metabolism. We uncover a complex network of positive interactions that buffer alterations in RNA degradation. We reveal important reciprocal actions between RNA decay and transcription and explore alleviating interactions between RNA splicing and DIS3 mediated degradation. We also use a large scale library of genes associated with RNA metabolism to determine genetic interactions of nuclear DIS3 and cytoplasmic DIS3L, revealing their unique functions in RNA degradation and uncovering cooperation between the cytoplasmic degradation and nuclear processing of RNA. Finally, genome-wide siRNA screening of DIS3 reveals processes such as microtubule organization and regulation of telomerase activity that are also functionally associated with nuclear exosome-mediated RNA degradation.

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Phosphoproteomic analysis of chondrocytes after short-term exposure to inorganic polyphosphate

10.1101/2021.07.30.454470 ◽

2021 ◽

Author(s):

Uros Kuzmanov ◽

Rahul Gawri ◽

Alena Zelinka ◽

Keith A Russel ◽

Shin-Haw Lee ◽

...

Keyword(s):

Large Scale ◽

Articular Chondrocytes ◽

Treatment Options ◽

Biological Molecules ◽

Inorganic Polyphosphate ◽

Naturally Occurring ◽

Molecular Events ◽

Short Term Exposure ◽

Matrix Production

Osteoarthritis is a debilitating disease of the joint that affects over 230 million people worldwide. Currently there are no treatments that slow the progression of this disease. For these reasons, new biological treatment options are currently being explored. Inorganic polyphosphates are naturally occurring biological molecules that have an anabolic effect on chondrocytes grown in vitro in the presence of Ca2+. We hypothesized that when examining significant changes in protein phosphorylation, key candidates would emerge that could help to elucidate the anabolic effects of polyphosphate on chondrocytes. Therefore, we conducted a large-scale quantitative proteomic and phosphoproteomic study of bovine primary articular chondrocytes after 30-minute treatment with inorganic polyphosphate and Ca2+. Mass spectrometry identified more than 6000 phosphorylation sites on ~1600 chondrocyte phosphoproteins while proteomic analysis detected approximately 4100 proteins. Analysis of the data revealed a swift and dynamic response to polyphosphate after 30 minutes. What emerged from the list of proteins most affected by the treatment were proteins with key roles in chondrogenesis including TNC, IGFBP-5, and CTGF, indicating that polyphosphate plays an important role in chondrocyte metabolism. This phosphoproteome serves as a meaningful resource to help elucidate the molecular events that contribute to extracellular matrix production in cartilage.

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Some comments about observations and image processing of comet 29P/Schwassmann-Wachmann 1

International Astronomical Union Colloquium ◽

10.1017/s0252921100031493 ◽

1999 ◽

Vol 173 ◽

pp. 243-248

Author(s):

D. Kubáček ◽

A. Galád ◽

A. Pravda

Keyword(s):

Image Processing ◽

Large Scale ◽

Harvard College ◽

Oak Ridge ◽

Large Scale Structures ◽

Short Period Comet ◽

Short Period ◽

Scale Structures ◽

Harvard College Observatory ◽

Period Comet

AbstractUnusual short-period comet 29P/Schwassmann-Wachmann 1 inspired many observers to explain its unpredictable outbursts. In this paper large scale structures and features from the inner part of the coma in time periods around outbursts are studied. CCD images were taken at Whipple Observatory, Mt. Hopkins, in 1989 and at Astronomical Observatory, Modra, from 1995 to 1998. Photographic plates of the comet were taken at Harvard College Observatory, Oak Ridge, from 1974 to 1982. The latter were digitized at first to apply the same techniques of image processing for optimizing the visibility of features in the coma during outbursts. Outbursts and coma structures show various shapes.

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Evolution of Large-Scale Coronal Structures

International Astronomical Union Colloquium ◽

10.1017/s0252921100024945 ◽

1994 ◽

Vol 144 ◽

pp. 29-33

Author(s):

P. Ambrož

Keyword(s):

Magnetic Field ◽

Field Line ◽

Large Scale ◽

Coronal Magnetic Field ◽

Source Surface ◽

Solar Cycles ◽

Characteristic Relationship ◽

The Magnetic Field ◽

Coronal Structures

AbstractThe large-scale coronal structures observed during the sporadically visible solar eclipses were compared with the numerically extrapolated field-line structures of coronal magnetic field. A characteristic relationship between the observed structures of coronal plasma and the magnetic field line configurations was determined. The long-term evolution of large scale coronal structures inferred from photospheric magnetic observations in the course of 11- and 22-year solar cycles is described.Some known parameters, such as the source surface radius, or coronal rotation rate are discussed and actually interpreted. A relation between the large-scale photospheric magnetic field evolution and the coronal structure rearrangement is demonstrated.

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Large-scale Motion of Solar Filaments

International Astronomical Union Colloquium ◽

10.1017/s0252921100064502 ◽

2000 ◽

Vol 179 ◽

pp. 205-208

Author(s):

Pavel Ambrož ◽

Alfred Schroll

Keyword(s):

Magnetic Flux ◽

Proper Motion ◽

Time Scale ◽

Large Scale ◽

Accuracy Of Measurements ◽

Solar Filaments ◽

General Movement ◽

Scale Motion ◽

Velocity Scatter

AbstractPrecise measurements of heliographic position of solar filaments were used for determination of the proper motion of solar filaments on the time-scale of days. The filaments have a tendency to make a shaking or waving of the external structure and to make a general movement of whole filament body, coinciding with the transport of the magnetic flux in the photosphere. The velocity scatter of individual measured points is about one order higher than the accuracy of measurements.

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Some applications of electron beam microanalysis techniques in VLSI process evaluation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010007463x ◽

1983 ◽

Vol 41 ◽

pp. 160-161

Author(s):

Simon Thomas

Keyword(s):

Integrated Circuits ◽

Process Evaluation ◽

Large Scale ◽

Technology Development ◽

Analytical Techniques ◽

Successful Implementation ◽

Analysis Of Failures ◽

Characterization Of Materials ◽

Circuits Vlsi

Trends in the technology development of very large scale integrated circuits (VLSI) have been in the direction of higher density of components with smaller dimensions. The scaling down of device dimensions has been not only laterally but also in depth. Such efforts in miniaturization bring with them new developments in materials and processing. Successful implementation of these efforts is, to a large extent, dependent on the proper understanding of the material properties, process technologies and reliability issues, through adequate analytical studies. The analytical instrumentation technology has, fortunately, kept pace with the basic requirements of devices with lateral dimensions in the micron/ submicron range and depths of the order of nonometers. Often, newer analytical techniques have emerged or the more conventional techniques have been adapted to meet the more stringent requirements. As such, a variety of analytical techniques are available today to aid an analyst in the efforts of VLSI process evaluation. Generally such analytical efforts are divided into the characterization of materials, evaluation of processing steps and the analysis of failures.

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Electron microscopy study of reactively sputter-deposited Ti/N films on silicon

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100131449 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1362-1363

Author(s):

V. C. Kannan ◽

A. K. Singh ◽

R. B. Irwin ◽

S. Chittipeddi ◽

F. D. Nkansah ◽

...

Keyword(s):

Integrated Circuits ◽

Large Scale ◽

Hexagonal Phase ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Process Conditions ◽

Tin Films ◽

Wide Range ◽

Fcc Phase ◽

Circuits Vlsi

Titanium nitride (TiN) films have historically been used as diffusion barrier between silicon and aluminum, as an adhesion layer for tungsten deposition and as an interconnect material etc. Recently, the role of TiN films as contact barriers in very large scale silicon integrated circuits (VLSI) has been extensively studied. TiN films have resistivities on the order of 20μ Ω-cm which is much lower than that of titanium (nearly 66μ Ω-cm). Deposited TiN films show resistivities which vary from 20 to 100μ Ω-cm depending upon the type of deposition and process conditions. TiNx is known to have a NaCl type crystal structure for a wide range of compositions. Change in color from metallic luster to gold reflects the stabilization of the TiNx (FCC) phase over the close packed Ti(N) hexagonal phase. It was found that TiN (1:1) ideal composition with the FCC (NaCl-type) structure gives the best electrical property.

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Evolution of the microstructure of Cu(Ti)/SiO2 layers annealed in NH3

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100138634 ◽

1995 ◽

Vol 53 ◽

pp. 452-453

Author(s):

J. Liu ◽

N. D. Theodore ◽

D. Adams ◽

S. Russell ◽

T. L. Alford ◽

...

Keyword(s):

Thermal Stability ◽

Titanium Nitride ◽

Large Scale ◽

Standard Procedure ◽

Alloy Film ◽

Electron Beam Evaporation ◽

Copper Metallization ◽

Nominal Composition ◽

Ti Alloy ◽

Thermally Grown

Copper-based metallization has recently attracted extensive research because of its potential application in ultra-large-scale integration (ULSI) of semiconductor devices. The feasibility of copper metallization is, however, limited due to its thermal stability issues. In order to utilize copper in metallization systems diffusion barriers such as titanium nitride and other refractory materials, have been employed to enhance the thermal stability of copper. Titanium nitride layers can be formed by annealing Cu(Ti) alloy film evaporated on thermally grown SiO2 substrates in an ammonia ambient. We report here the microstructural evolution of Cu(Ti)/SiO2 layers during annealing in NH3 flowing ambient.The Cu(Ti) films used in this experiment were prepared by electron beam evaporation onto thermally grown SiO2 substrates. The nominal composition of the Cu(Ti) alloy was Cu73Ti27. Thermal treatments were conducted in NH3 flowing ambient for 30 minutes at temperatures ranging from 450°C to 650°C. Cross-section TEM specimens were prepared by the standard procedure.

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