Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells.

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation

Collagen organization within the cartilage of TRPV4(-/-) mice studied with two-photon microscopy and polarized second harmonic generation

The polymodal channel TRPV4 has been shown to regulate development and maintenance of cartilage. Here we investigate whether TRPV4 activity regulates the early deposition and structure of collagen matrix in the femoral head cartilage by comparing the 3D morphology and the sub-micrometer organization of the collagen matrix between wild type and TRPV4(-/-) mice pups four to five days old. Two-photon microscopy can be used to conduct label-free imaging of cartilage, as collagen generates a second harmonic signal (second harmonic generation [SHG]) under pulsed infrared excitation. In one set of measurements, we use circularly polarized laser light to reconstruct the 3D morphology of the femoral head cartilage and to measure the tissue thickness. Second, by rotating the direction of the linearly polarized light and using polarized SHG detection, we investigate the sub-micrometer orientation of collagen fibers in the cartilage. At this developmental stage, we cannot detect statistically significant differences between the two mice strains, although a tendency toward a more random orientation of collagen fibers and a higher thickness of the whole cartilage seems to characterize the TRPV4(-/-) mice. We discuss possible reasons for these observations

Radiocarbon investigation of the Superlative African Baobabs from Savé Valley Conservancy, Zimbabwe

Author Posting. © Studia Chemia, 2019. Studia Universitatis Babes-Bolyai Seria Chemia is an Open Access Journal (read, download, copy, distribute, print for research use, search, or link to the full texts of articles). The definitive version was published in Studia Universitatis Babes-Bolyai, Seria Chemia , 64(2), Tom II, (2019): 411-419, doi:10.24193/subbchem.2019.2.35.The article reports the radiocarbon investigation results of the superlative African baobabs from Savé Valley, Zimbabwe. Several wood samples collected from these baobab were analysed by AMS (accelerator mass spectrometry) radiocarbon dating. The radiocarbon dates of the oldest samples were 1529 ± 14 BP for Matendere Big baobab, 1179 ± 19 BP for Chishakwe Big tree and 1096 ± 35 BP for Mokore Giant baobab. The corresponding calibrated ages are 1430 ± 15, 1090 ± 40 and 1020 ± 25 calendar yr. The oldest tree from Savé Valley, which we described previously, is the Humani Bedford Old baobab. The radiocarbon date of its oldest sample, 1655 ± 14 BP, corresponds to a calibrated age of 1580 ± 30 calendar yr.Authors would like to acknowledge Léon and Judy Duplessis, the owners of the Matendere Rach, Lisa-Jane Campbell of Chishakwe Ranch, Roger Whittall, the owner of the Humani Ranch and his wife Anne Whittall, Greg and Melanie Duckworth of Mokore Ranch for granting access in the ranches and for authorising the investigation and sampling of the monumental baobabs. The research was funded by the Romanian Ministry of National Education CNCS-UEFISCDI under grant PN-III-P4-ID-PCE-2016-0776, Nr. 90/2017

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements

An automated multiwell plate reading film microscope for live cell autofluorescence lifetime assays

Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine

Radiocarbon dating of a very large African baobab from Limpopo, South Africa : investigation of the Sagole Big Tree

Author Posting. © Studia Chemia, 2017. This article is posted here by permission of Studia Chemia for personal use, not for redistribution. The definitive version was published in Studia Universitatis Babes-Bolyai, Seria Chemia 62, no. 2, Tom 2 (2017): 355-364, doi:10.24193/subbchem.2017.2.28.The article reports the AMS (accelerator mass spectrometry) radiocarbon dating results of Sagole Big tree, a giant African baobab from Limpopo, South Africa. Several wood samples were collected from the walls of its inner cavity and dated by radiocarbon. The age values along the cavity samples increase with the distance into the wood. This anomaly shows that the cavity is a false one. The oldest sample segment had a radiocarbon date of 781 ± 29 BP, which corresponds to a calibrated age of 740 ± 15 yr. We estimate that the oldest part of the Sagole baobab has an age of 800-900 yr. We determined that the tree has a closed ring-shaped structure, which consists of a large unit with six fused stems and of two additional leaning stems.The research was funded by the Romanian Ministry of Scientific Research CNCSUEFISCDI under grant PN-II-ID-PCE-2013-76

Radiocarbon dating of a very old African baobab from Savé Valley, Zimbabwe

Author Posting. © Studia Chemia, 2016. This article is posted here by permission of Studia Chemia for personal use, not for redistribution. The definitive version was published in Studia Chemia 2016, no. 4 (2016): 7-20.The article reports the radiocarbon investigation results of the Humani Bedford baobab, an old African baobab from Savé Valley, Zimbabwe. Two wood samples were collected from the large inner cavity. Several segments were extracted from these samples and analysed by AMS (accelerator mass spectrometry) radiocarbon dating. We found that the age values of segments increase with the distance into the wood. This major anomaly is characteristic to multi-stemmed baobabs with a closed ring-shaped structure and a false cavity inside. The investigation of the Humani Bedford baobab evinced that the baobab consists of three fused stems. The fourth stem of the ring is missing. The oldest dated segment was found to have a radiocarbon date of 1655 ± 14 BP, which corresponds to a calibrated age of 1575 ± 30 yr. The dating results show that the stems which build the ring stopped growing toward the false cavity more than 600 yr ago. By considering the position of the oldest segment in the investigated stem, we concluded that the Humani Bedford baobab is around 1800 yr old. According to our dating results, the Humani Bedford baobab becomes the oldest living African baobab.The research was funded by the Romanian Ministry of National Education CNCS-UEFISCDI under grant PN-II-ID-PCE-2013-76

Age and growth rate dynamics of an old African baobab determined by radiocarbon dating

Author Posting. © Arizona Board of Regents on behalf of the University of Arizona, 2010. This article is posted here by permission of Dept. of Geosciences, University of Arizona for personal use, not for redistribution. The definitive version was published in Radiocarbon 52 (2010): 727-734.In 2008, a large African baobab (Adansonia digitata L.) from Makulu Makete, South Africa, split vertically into 2 sections, revealing a large enclosed cavity. Several wood samples collected from the cavity were processed and radiocarbon dated by accelerator mass spectrometry (AMS) for determining the age and growth rate dynamics of the tree. The 14C date of the oldest sample was found to be of 1016 ± 22 BP, which corresponds to a calibrated age of 1000 ± 15 yr. Thus, the Makulu Makete tree, which eventually collapsed to the ground and died, becomes the second oldest African baobab dated accurately to at least 1000 yr. The conventional growth rate of the trunk, estimated by the radial increase, declined gradually over its life cycle. However, the growth rate expressed more adequately by the cross-sectional area increase and by the volume increase accelerated up to the age of 650 yr and remained almost constant over the past 450 yr.This material is based on work supported by a grant from the Romanian National University Research Council (PN II - IDEI 2354 Nr. 1092) and by US National Science Foundation under Cooperative Agreement OCE-022828996

Radiocarbon investigation of the pedunculate oak of Botosana, Romania

Author Posting. © Studia Chemia, 2018. Studia Universitatis Babes-Bolyai Chemia is an Open Access Journal (read, download, copy, distribute, print for research use, search, or link to the full texts of articles). The definitive version was published in Studia Universitatis Babes-Bolyai Chemia 63(4), (2018): 7-13, doi: 10.24193/subbchem.2018.4.01.The article discloses the AMS (accelerator mass spectrometry) radiocarbon dating results of the pedunculate oak of Botoşana. Four wood samples were extracted from its trunk. Five segments extracted from these samples were analyzed by AMS radiocarbon. Their radiocarbon dates were found to be between 161 ± 21 BP and 260 ± 20 BP. These values correspond to calibrated ages of 235 – 365 years. The dating results extrapolated to the geometric center of the trunk indicate an age of 645 ± 50 years for the oak of Botoşana.The research was funded by the Romanian Ministry of Research and Innovation CNCS-UEFISCDI under grant PN-III-P4-ID-PCE-2016-0776, Nr. 90/2017