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Measuring the three-dimensional (3D) distribution of chemistry in nanoscale matter is a longstanding challenge for metrological science. The inelastic scattering events required for 3D chemical imaging are too rare, requiring high beam exposure that destroys the specimen before an experiment completes. Even larger doses are required to achieve high resolution. Thus, chemical mapping in 3D has been unachievable except at lower resolution with the most radiation-hard materials. Here, high-resolution 3D chemical imaging is achieved near or below one nanometer resolution in a Au-Fe3O4 metamaterial, Co3O4 - Mn3O4 core-shell nanocrystals, and ZnS-Cu0.64S0.36 nanomaterial using fused multi-modal electron tomography. Multi-modal data fusion enables high-resolution chemical tomography often with 99% less dose by linking information encoded within both elastic (HAADF) and inelastic (EDX / EELS) signals. Now sub-nanometer 3D resolution of chemistry is measurable for a broad class of geometrically and compositionally complex materials.

Time lapse video showing lipid droplet turnover in less motile cells away from the leading edge

5 Supplementary Figures and legends Legends for 2 supplementary movies

Time lapse video showing lipid droplet turnover in migratory leading-edge tumor cells

Oncogene-induced metabolic reprogramming is a hallmark of pancreatic cancer (PDAC), yet the metabolic drivers of metastasis are unclear. In PDAC, obesity and excess fatty acids accelerate tumor growth and increase metastasis. Here, we report that excess lipids, stored in organelles called lipid droplets (LD), are a key resource to fuel the energy-intensive process of metastasis. The oncogene KRAS controlled the storage and utilization of LD through regulation of hormone-sensitive lipase (HSL), which was downregulated in human PDAC. Disruption of the KRAS–HSL axis reduced lipid storage, reprogrammed tumor cell metabolism, and inhibited invasive migration in vitro and metastasis in vivo. Finally, microscopy-based metabolic analysis revealed that migratory cells selectively utilize oxidative metabolism during the process of migration to metabolize stored lipids and fuel invasive migration. Taken together, these results reveal a mechanism that can be targeted to attenuate PDAC metastasis.Significance:KRAS-dependent regulation of HSL biases cells towards lipid storage for subsequent utilization during invasion of pancreatic cancer cells, representing a potential target for therapeutic intervention.See related commentary by Man et al., p. 4886

BACKGROUND/AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography–tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3(+) T cells (G9a-inibitors–T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors–Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid–induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer–induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2-interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.

Technological opportunities are explored to enhance detection schemes in transmission electron microscopy (TEM) that build on the detection of single-electron scattering events across the typical spectrum of interdisciplinary applications. They range from imaging with high spatiotemporal resolution to diffraction experiments at the window to quantum mechanics, where the wave-particle dualism of single electrons is evident. At the ultimate detection limit, where isolated electrons are delivered to interact with solids, we find that the beam current dominates damage processes instead of the deposited electron charge, which can be exploited to modify electron beam-induced sample alterations. The results are explained by assuming that all electron scattering are inelastic and include phonon excitation that can hardly be distinguished from elastic electron scattering. Consequently, a coherence length and a related coherence time exist that reflect the interaction of the electron with the sample and change linearly with energy loss. Phonon excitations are of small energy (

Guerbet alcohols, a class of β-branched terminal alcohols, find widespread application because of their low melting points and excellent fluidity. Because of the limitations in the activity and selectivity of existing Guerbet catalysts, Guerbet alcohols are not currently produced via the Guerbet reaction but via hydroformylation of oil-derived alkenes followed by aldol condensation. In pursuit of a one-step synthesis of Guerbet alcohols from simple linear alcohol precursors, we show that MOF-derived RuCo alloys achieve over a million turnovers in the Guerbet reaction of 1-propanol, 1-butanol, and 1-pentanol. The active catalyst is formed in situ from ruthenium-impregnated metal-organic framework MFU-1. XPS and XAS studies indicate that the precatalyst is composed of Ru precursor trapped inside the MOF pores with no change in the oxidation state or coordination environment of Ru upon MOF incorporation. The significantly higher reactivity of Ru-impregnated MOF versus a physical mixture of Ru precursor and MOF suggests that the MOF plays an important role in templating the formation of the active catalyst and/or its stabilization. XPS reveals partial reduction of both ruthenium and MOF-derived cobalt under the Guerbet reaction conditions, and TEM/EDX imaging shows that Ru is decorated on the edges of dense nanoparticles, as well as thin nanoplates of CoOx. The use of ethanol rather than higher alcohols as a substrate results in lower turnover frequencies, and RuCo recovered from ethanol upgrading lacks nanostructures with plate-like morphology and does not exhibit Ru-enrichment on the surface and edge sites. Notably, 1H and 31P NMR studies show that through use of K3PO4 as a base promoter in the RuCo-catalyzed alcohol upgrading, the formation of carboxylate salts, a common side product in the Guerbet reaction, was effectively eliminated.

High-throughput grain mapping with sub-nanometer spatial resolution is demonstrated using scanning nanobeam electron diffraction (also known as 4D scanning transmission electron microscopy, or 4D-STEM) combined with high-speed direct-electron detection. An electron probe size down to 0.5 nm in diameter is used and the sample investigated is a gold–palladium nanoparticle catalyst. Computational analysis of the 4D-STEM data sets is performed using a disk registration algorithm to identify the diffraction peaks followed by feature learning to map the individual grains. Two unsupervised feature learning techniques are compared: principal component analysis (PCA) and non-negative matrix factorization (NNMF). The characteristics of the PCA versus NNMF output are compared and the potential of the 4D-STEM approach for statistical analysis of grain orientations at high spatial resolution is discussed.