Notes

Your (not)discovering of ecology by Ak Indigenous ecologists.
Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. RP-3500 ic50 This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.Mass cytometry (MC) is a bioanalytical technique that uses metal-tagged antibodies (Abs) for high-dimensional single-cell immunoassays. Currently, this technology can measure over 40 parameters simultaneously on individual cells using metal-chelating polymer (MCP) based reagents. However, MC can in principle detect up to 135 parameters with the development of new elemental mass tags. Here we report the development of a tantalum oxide nanoparticle (NP)-based mass tag for MC immunoassays. Uniform-sized amine-functionalized tantalum oxide NPs (d ∼ 5.7 nm) were synthesized via a one-pot two-step reverse microemulsion method. These amine-functionalized NPs were further modified with azide groups by reacting with azide-PEG2k succinimidyl carboxymethyl ester (NHS-PEG2k-N3) cross-linkers. The Ab-NP conjugates were prepared by reacting azide-functionalized NPs with dibenzocyclooctyne (DBCO)-functionalized primary or secondary Abs (DBCO-Ab) followed by fast protein size exclusion liquid chromatography (FPLC) purification. Three Ab-NP conjugates (TaO2-PEG2k-goat antimouse, TaO2-PEG2k-CD25, TaO2-PEG2k-CD196) were fabricated and tested in MC immunoassays. For the TaO2-PEG2k-goat antimouse conjugate, we showed that it can effectively detect abundant CD20 biomarkers on Ramos cells. For TaO2-PEG2k-CD25 and TaO2-PEG2k-CD196 conjugates, we demonstrated that these Ab-NP conjugates could be integrated into the commercial Ab staining panels for high-dimensional single-cell immune profiling of human peripheral blood mononuclear cells.Membraneless organelles formed by liquid-liquid phase separation are dynamic structures that are employed by cells to spatiotemporally regulate their interior. Indeed, complex coacervation-based phase separation is involved in a multitude of biological tasks ranging from photosynthesis to cell division to chromatin organization, and more. Here, we use an on-chip microfluidic method to control and study the formation of membraneless organelles within liposomes, using pH as the main control parameter. We show that a transmembrane proton flux that is created by a stepwise change in the external pH can readily bring about the coacervation of encapsulated components in a controlled manner. We employ this strategy to induce and study electrostatic as well as hydrophobic interactions between the coacervate and the lipid membrane. Electrostatic interactions using charged lipids efficiently recruit coacervates to the membrane and restrict their movement along the inner leaflet. Hydrophobic interactions via cholesterol-tagged RNA molecules provide even stronger interactions, causing coacervates to wet the membrane and affect the local lipid-membrane structure, reminiscent of coacervate-membrane interactions in cells. The presented technique of pH-triggered coacervation within cell-sized liposomes may find applications in synthetic cells and in studying biologically relevant phase separation reactions in a bottom-up manner.ConspectusAlkyl and alkenyl arenes are of substantial value in both large-scale and fine chemical processes. Billions of pounds of alkyl and alkenyl arenes are produced annually. Historically, the dominant method for synthesis of alkyl arenes is acid-catalyzed arene alkylation, and alkenyl arenes are often synthesized in a subsequent dehydrogenation step. But these methods have limitations that result from the catalytic mechanism including (1) common polyalkylation, which requires an energy intensive transalkylation process, (2) quantitative selectivity for Markovnikov products for arene alkylation using α-olefins, (3) for substituted arenes, regioselectivity that is dictated by the electronic character of the arene substituents, (4) inability to form alkenyl arenes in a single process, and (5) commonly observed slow reactivity with electron-deficient arenes. Transition-metal-catalyzed aryl-carbon coupling reactions can produce alkyl or alkenyl arenes from aryl halides. However, these reactions often generate products when using substituted olefins. Finally, when using monosubstituted arenes, consistent with a metal-mediated C-H activation process, Rh-catalyzed alkenylation of substituted arenes shows selectivity for meta- and para-alkenylation products.The polyene amphotericin B (AMB) exerts a powerful and broad antifungal activity. AMB acts by (i) binding to ergosterol, leading to pore formation at the fungal plasma membrane with subsequent ion leakage, and (ii) inducing the intracellular accumulation of reactive oxygen species (ROS). Herein, we have deciphered the AMB resistance mechanisms in clinical isolates of Candida haemulonii complex (C. haemulonii, C. duobushaemulonii, C. haemulonii var. vulnera) in comparison to other clinically relevant non-albicans Candida species. Membrane gas chromatography-mass spectrometry analysis revealed that the vast majority of sterols were composed of ergosterol pathway intermediates, evidencing the absence of AMB target. Supporting this data, C. haemulonii species complex demonstrated poor membrane permeability after AMB treatment. Regarding the oxidative burst, AMB induced the formation of ROS in all species tested; however, this phenomenon was slightly seen in C. haemulonii complex isolates. Our results indicated that these isolates displayed altered respiratory status, as revealed by their poor growth in nonfermented carbon sources, low consumption of oxygen, and derisive mitochondrial membrane potential.
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