| 1. |
- Bart, Genevieve, et al.
(author)
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Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo-and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3
- 2015
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:18, s. 11479-11490
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Journal article (peer-reviewed)abstract
- In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.
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| 2. |
- Datta, Timir, et al.
(author)
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Integration of CMOS Chips into LOCs for Cell-Based Sensing
- 2014
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In: Proceedings of Biosensors 2014, Australia.
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Conference paper (peer-reviewed)abstract
- Incorporating complementary metal oxide semiconductor (CMOS) chips that can perform signal processing, control, information readout, and direct sensing into microfluidic systems adds powerful capabilities to lab on a chip (LOC) devices. For example, on-chip sensors allow system miniaturization, amplifiers placed directly under the sensors provide high signal to noise ratios (SNRs), and signal processing circuitry reduces the amount of data that must be communicated off-chip. Packaging such chips to expose the sensors on the surface to a fluid environment while protecting the input/output region at the periphery has been challenging, however. We present a new packaging method based on forming an epoxy handle wafer around the chip, photolithographic patterning of metal and polymer films for interconnection and passivation, and bonding to PDMS microfluidics. Such packaged chips last for months in the incubator and can be sterilized and re-used. We will show two examples of cell-based sensing with these systems using chips produced in a commercially-available CMOS technology: monitoring the cytotoxicity of nanomaterials through capacitance changes and recording action potentials from electrogenic cells. Adherent cells normally spread out on surfaces, while stressed cells contract and apoptosis leads to detachment. A chip was produced consisting of an array of fully differential capacitance sensors and readout circuitry. Cells (kidney, Cercopithecus aethiops) were cultured on the chip surface to confluence and then exposed to cytotoxic TiO2 nanowires. Cell viability was evaluated with both the chip and a commercial cytotoxicity kit. Preliminary results indicate that viability can be monitored by capacitance measurements. In the second example, a cluster of cardiomyocytes was cultured on the surface of a different chip having an array of electrodes connected to on-chip amplifiers. Electrical recordings showed strong action potentials from the cluster, corresponding in time with the beating of the clump. The signal amplitude decreased with distance to the electrodes, as expected
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| 3. |
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| 4. |
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| 5. |
- Halonen, Niina, et al.
(author)
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Low temperature co-fired ceramic package for lab-on-CMOS applied in cell viability monitoring
- 2015
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In: Eurosensors 2015. - : Elsevier BV. ; , s. 1079-1082
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Conference paper (peer-reviewed)abstract
- Lab-on-CMOS chips (LOCMOS) are sophisticated miniaturized analysis tools based on integrated circuit (IC) microchips performing various laboratory functions. We have developed a low temperature co-fired ceramic (LTCC) package for a LOCMOS application regarding cytotoxicity assessment of nanomaterials. The LTCC packaged capacitance sensor chip is designed for long-term cell viability monitoring during nanoparticle exposure. The introduced LTCC package utilizes the flip chip bonding technique, and it is biocompatible as well as able to withstand the environmental conditions required to maintain mammalian cell culture directly on the surface of a complementary metal oxide semiconductor (CMOS) integrated circuit.
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| 6. |
- Halonen, Niina, et al.
(author)
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Low temperature co-fired ceramic packaging of CMOS capacitive sensor chip towards cell viability monitoring
- 2016
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In: Beilstein Journal of Nanotechnology. - : BEILSTEIN-INSTITUT. - 2190-4286. ; 7, s. 1871-1877
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Journal article (peer-reviewed)abstract
- Cell viability monitoring is an important part of biosafety evaluation for the detection of toxic effects on cells caused by nanomaterials, preferably by label-free, noninvasive, fast, and cost effective methods. These requirements can be met by monitoring cell viability with a capacitance-sensing integrated circuit (IC) microchip. The capacitance provides a measurement of the surface attachment of adherent cells as an indication of their health status. However, the moist, warm, and corrosive biological environment requires reliable packaging of the sensor chip. In this work, a second generation of low temperature co-fired ceramic (LTCC) technology was combined with flip-chip bonding to provide a durable package compatible with cell culture. The LTCC-packaged sensor chip was integrated with a printed circuit board, data acquisition device, and measurement-controlling software. The packaged sensor chip functioned well in the presence of cell medium and cells, with output voltages depending on the medium above the capacitors. Moreover, the manufacturing of microfluidic channels in the LTCC package was demonstrated.
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| 7. |
- Kilpijarvi, Joni, et al.
(author)
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LTCC Packaged Ring Oscillator Based Sensor for Evaluation of Cell Proliferation
- 2018
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In: Sensors. - : MDPI. - 1424-8220. ; 18:10
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Journal article (peer-reviewed)abstract
- A complementary metal-oxide-semiconductor (CMOS) chip biosensor was developed for cell viability monitoring based on an array of capacitance sensors utilizing a ring oscillator. The chip was packaged in a low temperature co-fired ceramic (LTCC) module with a flip chip bonding technique. A microcontroller operates the chip, while the whole measurement system was controlled by PC. The developed biosensor was applied for measurement of the proliferation stage of adherent cells where the sensor response depends on the ratio between healthy, viable and multiplying cells, which adhere onto the chip surface, and necrotic or apoptotic cells, which detach from the chip surface. This change in cellular adhesion caused a change in the effective permittivity in the vicinity of the sensor element, which was sensed as a change in oscillation frequency of the ring oscillator. The sensor was tested with human lung epithelial cells (BEAS-2B) during cell addition, proliferation and migration, and finally detachment induced by trypsin protease treatment. The difference in sensor response with and without cells was measured as a frequency shift in the scale of 1.1 MHz from the base frequency of 57.2 MHz. Moreover, the number of cells in the sensor vicinity was directly proportional to the frequency shift.
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| 8. |
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| 9. |
- Missaghian, Parisa, et al.
(author)
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A dominant negative splice variant of the heparan sulfate biosynthesis enzyme NDST1 reduces heparan sulfate sulfation
- 2022
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In: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 32:6, s. 518-528
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Journal article (peer-reviewed)abstract
- NDST1 (glucosaminyl N-deacetylase/N-sulfotransferase) is a key enzyme in heparan sulfate (HS) biosynthesis, where it is responsible for HS N-deacetylation and N-sulfation. In addition to the full length human enzyme of 882 amino acids, here designated NDST1A, a shorter form containing 825 amino acids (NDST1B) is synthesized after alternative splicing of the NDST1 mRNA. NDST1B is mostly expressed at a low level, but increased amounts are seen in several types of cancer where it is associated with shorter survival. In this study, we aimed at characterizing the enzymatic properties of NDST1B and its effect on HS biosynthesis. Purified recombinant NDST1B lacked both N-deacetylase and N-sulfotransferase activities. Interestingly, HEK293 cells overexpressing NDST1B synthesized HS with reduced sulfation and altered domain structure. Fluorescence resonance energy transfer-microscopy demonstrated that both NDST1A and NDST1B had the capacity to interact with the HS copolymerase subunits EXT1 and EXT2 and also to form NDST1A/NDST1B dimers. Since lysates from cells overexpressing NDST1B contained less NDST enzyme activity than control cells, we suggest that NDST1B works in a dominant negative manner, tentatively by replacing the active endogenous NDST1 in the enzyme complexes taking part in biosynthesis.
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| 10. |
- Tykesson, Emil, et al.
(author)
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Dermatan sulfate epimerase 1 and dermatan 4-O-sulfotransferase 1 form complexes that generate long epimerized 4-O-sulfated blocks
- 2018
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In: Journal of Biological Chemistry. - 0021-9258. ; 293:35, s. 13725-13735
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Journal article (peer-reviewed)abstract
- During the biosynthesis of chondroitin/dermatan sulfate (CS/ DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid– containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid–forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.
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