THROUGH-BOND ENERGY TRANSFER CASSETTES FOR MULTIPLEXING & DEVELOPMENT OF METHODS FOR PROTEIN MONO-LABELING

A set of three through-bond energy transfer cassettes based on BODIPY as a donor and cyanine dyes as acceptors has been prepared via Sonogashira couplings, and their photophysical properties were examined. These cassettes fluoresce around 600 to 800 nm and are resolved by approximately 100 nm. This property is an important factor for multiplexing study in cellular imaging. Several useful fluorescent probes such as 5- and 6-carboxyfluorescein, water-soluble BODIPY, and water-soluble Nile Blue dyes, have also been synthesized and their photophysical properties studied. We have also attempted to develop a method for protein mono-labeling via a solidphase approach. The labeling of protein with one fluorescent dye facilitates quantification and single molecule imaging in biological applications. Various solidsupports such as PEGA, CPG, and BSA-coated CPG, were tested. Photolabile and chemically cleavable linkers were prepared to connect solid-supports and fluorophores. Unfortunately, our approach to the fluorescent mono-labeling of native proteins did not give us any conclusive results

Encapsulated Energy-transfer Cassettes With Extremely Well Resolved Fluorescent Outputs

This paper concerns the development of water-compatible fluorescent imaging probes with tunable photonic properties that can be excited at a single wavelength. Bichromophoric cassettes 1a-1c consisting of a BODIPY donor and a cyanine acceptor were prepared using a simple synthetic route, and their photophysical properties were investigated. Upon excitation of the BODIPY moiety at 488 nm the excitation energy is transferred through an acetylene bridge to the cyanine dye acceptor, which emits light at approximately 600, 700, and 800 nm, i.e., with remarkable dispersions. This effect is facilitated by efficient energy transfer that gives a quasi-Stokes shift between 86 and 290 nm, opening a huge spectral window for imaging. The emissive properties of the cassettes depend on the energy-transfer (ET) mechanism: the faster the transfer, the more efficient it is. Measurements of rates of ET indicate that a through-bond ET takes place in the cassettes la and 1b that is 2 orders of magnitude faster than the classical through-space, Forster ET. In the case of cassette 1c, however, both mechanisms are possible, and the rate measurements do not allow us to discern between them. Thus, the cassettes 1a-1c are well suited for multiplexing experiments in biotechnological methods that involve a single laser excitation source. However, for widespread application of these probes, their solubility in aqueous media must be improved. Consequently, the probes were encapsulated in calcium phosphate/silicate nanoparticles (diameter ca. 22 nm) that are freely dispersible in water. This encapsulation process resulted in only minor changes in the photophysical properties of the cassettes. The system based on cassette la was chosen to probe how effectively these nanoparticles could be used to deliver the dyes into cells. Encapsulated cassette la permeated Clone 9 rat liver cells, where it localized in the mitochondria and fluoresced through the acceptor part, i.e., red. Overall, this paper reports readily accessible, cyanine-based through-bond ET cassettes that are lypophilic but can be encapsulated to form nanoparticles that disperse freely in water. These particles can be used to enter cells and to label organelles

Multiple Sulfur Isotope Records of the 3.22 Ga Moodies Group, Barberton Greenstone Belt

Co-auteur étrangerInternational audienceThe Moodies Group, the uppermost unit in the Barberton Greenstone Belt (BGB) in SouthAfrica, is a ~3.7-km-thick coarse clastic succession accumulated on terrestrial-to-shallow marinesettings at around 3.22 Ga. The multiple sulfur isotopic composition of pyrite of Moodies intervalswas newly obtained to examine the influence of these depositional settings on the sulfur isotope record.Conglomerate and sandstone rocks were collected from three synclines north of the Inyoka Fault of thecentral BGB, namely, the Eureka, Dycedale, and Saddleback synclines. The sulfur isotopic compositionof pyrite was analyzed by Secondary Ion Mass Spectrometry (SIMS) for 6 samples from the threesynclines and by Isotope Ratio Mass Spectrometry (IR-MS) for 17 samples from a stratigraphic sectionin the Saddleback Syncline. The present results show a signal of mass-independent fractionation ofsulfur isotopes (S-MIF), although t-tests statistically demonstrated that the Moodies S-MIF signals(mostly 0% < D33S < +0.5%) are significantly small compared to the signal of the older Paleoarchean(3.6–3.2 Ga) records. These peculiar signatures might be related to initial deposition of detrital pyriteof juvenile origin from the surrounding intrusive (tonalite–trondhjemite–granodiorite; TTG) andfelsic volcanic rocks, and/or to secondary addition of hydrothermal sulfur during late metasomatism.Moreover, fast accumulation (~0.1–1 mm/year) of the Moodies sediments might have led to a reducedaccumulation of sulfur derived from an atmospheric source during their deposition. As a result, thesulfur isotopic composition of the sediments may have become susceptible to the secondary additionof metasomatic sulfur on a mass balance point of view. The sulfur isotopic composition of Moodiespyrite is similar to the composition of sulfides from nearby gold mines. It suggests that, after theMoodies deposition, metasomatic pyrite formation commonly occurred north of the Inyoka Fault inthe central BGB at 3.1–3.0 Ga

Up-regulation of microRNA-21 correlates with lower kidney cancer survival.

BackgroundMicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically.Methods and resultsWe measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation.ConclusionsThe current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21

Hydrogenation reactions of carbon on Earth: linking methane, margarine, and life

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in McGlynn, S. E., Glass, J. B., Johnson-Finn, K., Klein, F., Sanden, S. A., Schrenk, M. O., Ueno, Y., & Vitale-Brovarone, A. Hydrogenation reactions of carbon on Earth: linking methane, margarine, and life. American Mineralogist, 105(5), (2020): 599-608, doi:10.2138/am-2020-6928CCBYNCND.Hydrogenation reactions are a major route of electron and proton flow on Earth. Interfacing geology and organic chemistry, hydrogenations occupy pivotal points in the Earth’s global geochemical cycles. Some examples of hydrogenation reactions on Earth today include the production and consumption of methane in both abiotic and biotic reactions, the reduction of protons in hydrothermal settings, and the biological synthesis and degradation of fatty acids. Hydrogenation reactions were likely important for prebiotic chemistry on the early Earth, and today serve as one of the fundamental reaction classes that enable cellular life to construct biomolecules. An understanding and awareness of hydrogenation reactions is helpful for comprehending the larger web of molecular and material inter-conversions on our planet. In this brief review we detail some important hydrogenation and dehydrogenation reactions as they relate to geology, biology, industry, and atmospheric chemistry. Such reactions have implications ranging from the suite of reactions on early Earth to industrial applications like the production of hydrocarbon fuel.S.E.M. is supported by NSF Award 1724300 and JSPS KAKENHI Grant JP18H01325. A.V.B. is supported by ANR T-ERC, CNRS INSU-SYSTER, and Rita Levi Montalcini by MIUR. J.B.G. is supported by NASA Exobiology Grant NNX14AJ87G and 80NSSC19K0477. F.K. is supported by NSF-OCE award 1634032 and 1427274. M.O.S. is supported by the NASA Astrobiology Institute Rock-Powered Life Grant NNA15BB02A

Thermophilic Lithotrophy and Phototrophy in an Intertidal, Iron-rich, Geothermal Spring

Hydrothermal systems, including terrestrial hot springs, contain diverse and systematic arrays of geochemical conditions that vary over short spatial scales due to progressive interaction between the reducing hydrothermal fluids, the oxygenated atmosphere, and in some cases seawater. At Jinata Onsen, on Shikinejima Island, Japan, an intertidal, anoxic, iron- and hydrogen-rich hot spring mixes with the oxygenated atmosphere and sulfate-rich seawater over short spatial scales, creating an enormous range of redox environments over a distance ~10 m. We characterized the geochemical conditions along the outflow of Jinata Onsen as well as the microbial communities present in biofilms, mats, and mineral crusts along its traverse via 16S amplicon and shotgun metagenomic sequencing. The microbial community changed significantly downstream as temperatures and dissolved iron concentrations dropped and dissolved oxygen rose. Near the spring source, primary productivity appears limited, and is fueled primarily by oxidation of ferrous iron and molecular hydrogen by members of the Zetaproteobacteria and Aquificae, while downstream the microbial community is dominated by oxygenic Cyanobacteria. At Jinata Onsen, Cyanobacteria are abundant and productive even at ferrous iron concentrations of ~150 μM, which challenges the idea that iron toxicity limited cyanobacterial expansion in the Precambrian oceans. Several novel lineages of Bacteria are also present at Jinata Onsen, including previously uncharacterized members of the Chloroflexi and Caldithrichaeota phyla, positioning Jinata Onsen as a valuable site for future characterization of these clades

Geochemical and metagenomic characterization of Jinata Onsen, a Proterozoic-analog hot spring, reveals novel microbial diversity including iron-tolerant phototrophs and thermophilic lithotrophs

Hydrothermal systems, including terrestrial hot springs, contain diverse geochemical conditions that vary over short spatial scales due to progressive interactions between reducing hydrothermal fluids, the oxygenated atmosphere, and, in some cases, seawater. At Jinata Onsen on Shikinejima Island, Japan, an intertidal, anoxic, iron-rich hot spring mixes with the oxygenated atmosphere and seawater over short spatial scales, creating diverse chemical potentials and redox pairs over a distance of ~10 m. We characterized geochemical conditions along the outflow of Jinata Onsen as well as the microbial communities present in biofilms, mats, and mineral crusts along its traverse using 16S rRNA gene amplicon and genome-resolved shotgun metagenomic sequencing. Microbial communities significantly changed downstream as temperatures and dissolved iron concentrations decreased and dissolved oxygen increased. Biomass was more limited near the spring source than downstream, and primary productivity appeared to be fueled by the oxidation of ferrous iron and molecular hydrogen by members of Zetaproteobacteria and Aquificae. The microbial community downstream was dominated by oxygenic Cyanobacteria. Cyanobacteria are abundant and active even at ferrous iron concentrations of ~150 μM, which challenges the idea that iron toxicity limited cyanobacterial expansion in Precambrian oceans. Several novel lineages of Bacteria are also present at Jinata Onsen, including previously uncharacterized members of the phyla Chloroflexi and Calditrichaeota, positioning Jinata Onsen as a valuable site for the future characterization of these clades