Extracellular vesicles (EVs), naturally occurring nanosized vesicles secreted from cells, are essential for intercellular communication. They carry unique biomolecules on the surface or interior that are of great interest as biomarkers for various pathological conditions such as cancer. In this work, we use highresolution atomic force microscopy (AFM) and spectroscopy (AFS) techniques to demonstrate differences between EVs derived from colon cancer cells and colon epithelial cells at the singlevesicle level. We observe that EV populations are significantly increased in the cancer cell media compared to the normal cell EVs. We show that both EVs display an EV marker, CD9, while EVs derived from the cancer cells are slightly higher in density. Hyaluronan (HA) is a nonsulfated glycosaminoglycan linked to malignant tumor growth according to recent reports. Interestingly, at the single-vesicle level, colon cancer EVs exhibit significantly increased HA surface densities compared to the normal EVs. Spectroscopic measurements such as Fourier transform infrared (FT-IR), circular dichroism (CD), and Raman spectroscopy unequivocally support the AFM and AFS measurements. To our knowledge, it represents the first report of detecting HA-coated EVs as a potential colon cancer biomarker. Taken together, this sensitive approach will be useful in identifying biomarkers in the early stages of detection and evaluation of cancer.
The protein phosphatase calcineurin is activated in response to rising intracellular Ca 2+ levels and impacts fundamental cellular processes in organisms ranging from yeast to humans. In fungi, calcineurin orchestrates cellular adaptation to diverse environmental challenges and is essential for virulence of pathogenic species. To enable rapid and large-scale assessment of calcineurin activity in living, unperturbed yeast cells, we have generated stable and destabilized GFP transcriptional reporters under the control of a calcineurin-dependent response element (CDRE). Using the reporters, we show that the rapid dynamics of calcineurin activation and deactivation can be followed by flow cytometry and fluorescence microscopy. This system is compatible with live/dead staining that excludes confounding dead cells from the analysis. The reporters provide technology to monitor calcineurin dynamics during stress and ageing and may serve as a drug-screening platform to identify novel antifungal compounds that selectively target calcineurin.-catalytic subunit of CN, CnB -regulatory subunit of CN, GFP -green fluorescent protein, PI -propidium iodide. J. Diessl et al. (2020) Calcineurin activity in living yeast OPEN ACCESS | www.microbialcell.com 107 Microbial Cell | APRIL 2020 | Vol. 7 No. 4 FIGURE 1: GFP and GFP PEST function as reporters of calcineurin activity. (A) Schematics of pAMS366-4XCDRE-lacZ, pAMS366-4xCDRE-GFP and pAMS366-4xCDRE-GFP PEST plasmids encoding reporters for CN activity. (B) CN activity was determined via β-gal activity or via flow cytometric quantification of GFP fluorescence intensities in exponentially growing wild type and ∆crz1 cells equipped with either pAMS366-4xCDRE-lacZ, pAMS366-4xCDRE-GFP or pAMS366-4xCDRE-GFP PEST . Values are shown as fold of ∆crz1 cells. Means ± SEM, n = 4. (C) CN activity was measured as in (B) in exponentially growing wild type, ∆pmr1, ∆cnb1 and ∆crz1 cells equipped with the indicated reporter plasmid.For stimulation of CN activity, cells were treated with 50 mM Ca 2+ 1 h prior to measurement. Fold of untreated wild type cells is shown. Dead cells were excluded from the analysis via propidium iodide (PI) staining. Means ± SEM, n = 4. (D) Representative immunoblots of protein extracts from cells described in (C). Immunoblots were decorated with antibodies against β-gal or GFP, respectively, and Pgk1 as loading control. (E-G) Histograms of cells quantified in (C) indicating the shift in green fluorescence intensity of wild type cells with and without 50 mM Ca 2+ treatment and ∆pmr1 cells equipped with the GFP (E) or the GFP PEST reporter (F, G).
A void-enriched and highly strained porous Au−Ag nanoalloy (NP alloy ) has been synthesized from Au−Ag core−shell nanostructure by employing a galvanic replacement reaction and introducing Kirkendall voids in it. Obtained NP alloy acts both as an efficient cathodic and anodic material for methanol, ethylene glycol, and glycerol oxidation as well as oxygen reduction reactions simultaneously in an alkaline medium. High catalytic efficacy (low onset potential (E on ) and high current density (j)), wide thermal stability, and positive alcohol tolerance response of NP alloy sets them as a practical bifunctional electrode coating alternative compared to Pt/C in designing an efficient alkaline direct alcohol fuel cell.
Overexposure to manganese disrupts cellular energy metabolism across species, but the molecular mechanism underlying manganese toxicity remains enigmatic. Here, we report that excess cellular manganese selectively disrupts coenzyme Q (CoQ) biosynthesis, resulting in failure of mitochondrial bioenergetics. While respiratory chain complexes remain intact, the lack of CoQ as lipophilic electron carrier precludes oxidative phosphorylation and leads to premature cell and organismal death. At a molecular level, manganese overload causes mismetallation and proteolytic degradation of Coq7, a diiron hydroxylase that catalyzes the penultimate step in CoQ biosynthesis. Coq7 overexpression or supplementation with a CoQ headgroup analog that bypasses Coq7 function fully corrects electron transport, thus restoring respiration and viability. We uncover a unique sensitivity of a diiron enzyme to mismetallation and define the molecular mechanism for manganese-induced bioenergetic failure that is conserved across species.
Spherical gold nanoseed (∼5–6 nm)-induced (but not seed-mediated) silver nanorods (Hy-Au@AgNRs) of variable lengths have been synthesized by a new methodology that shows enhancement in catalytic activity as a function of nanorod length. Detailed characterization by atomic-scale resolution spectroscopy, precision scattering measurements, high-resolution microscopy, and theoretical modeling through the density functional theory (DFT) quantifies the presence of an enhanced number of multiple coaxial twin boundaries for longer Hy-Au@AgNRs, which ultimately results in an increased mechanical strain. By considering greater mechanical strain within Hy-Au@AgNRs, the density of states (DOS) calculation shows a prominent shift in electron density toward the Fermi level to assist in the tremendous catalytic activity of the longest nanorod (NR) (Hy-Au@AgNR840). Further assembling of these inherently active Hy-Au@AgNR840s by thiol click chemistry not only efficiently creates multiple low-coordinated crystal sites to improve their catalytic activity but also the resultant uniform two-dimensional (2D) platform shows better adsorptivity and easy moldability on the electrode surface for increased shelf life, a uniform porous structure to trap a large extent of redox systems, enhanced stability in a broad pH and solvent range to increase the applicability, and long-term stability under ambient conditions for safe storing, making this material a unique nonenzymatic scalable universal electrocatalytic platform. The ability of this material to act as a nonenzymatic universal catalytic platform has been verified by applying it for highly specific and ultrasensitive detection of a series of human metabolites, which include different important vitamins, potent endogenous antioxidants, essential amino acids for the biosynthesis of proteins, simple monosaccharides, and essential trace-metal ions. Our study for the first time mechanistically explores the combined role of anisometric seeding to create an intermetallic twin boundary along with its size to control the strain-induced catalytic activity to offer us a universal 2D electrocatalytic sensing platform by a combined approach of experiment and theory.
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