Sialylated N-glycans mediate monocyte uptake of extracellular vesicles secreted from Plasmodium falciparum-infected red blood cells

Glycoconjugates on extracellular vesicles (EVs) play a vital role in internalization and mediate interaction as well as regulation of the host immune system by viruses, bacteria, and parasites. During their intraerythrocytic life-cycle stages, malaria parasites, Plasmodium falciparum (Pf) mediate the secretion of EVs by infected red blood cells (RBCs) that carry a diverse range of parasitic and host-derived molecules. These molecules facilitate parasite-parasite and parasite-host interactions to ensure parasite survival. To date, the number of identified Pf genes associated with glycan synthesis and the repertoire of expressed glycoconjugates is relatively low. Moreover, the role of Pf glycans in pathogenesis is mostly unclear and poorly understood. As a result, the expression of glycoconjugates on Pf-derived EVs or their involvement in the parasite life-cycle has yet to be reported. Herein, we show that EVs secreted by Pf-infected RBCs carry significantly higher sialylated complex N-glycans than EVs derived from healthy RBCs. Furthermore, we reveal that EV uptake by host monocytes depends on N-glycoproteins and demonstrate that terminal sialic acid on the N-glycans is essential for uptake by human monocytes. Our results provide the first evidence that Pf exploits host sialylated N-glycans to mediate EV uptake by the human immune system cells

Observing electrochemical reactions on suspended graphene: an operando Kelvin probe force microscopy approach

An electrochemical micro-reactor sealed with a single-layer graphene (SLG) membrane is demonstrated that allows straightforward measurement with established scanning probe microscopies. SLG serves as a working electrode which separates the liquid electrochemical environment from the ambient to enable direct energy-level determination. Kelvin probe force microscopy (KPFM) thereby reveals the shifts in Fermi-level of suspended SLG under electrochemical reaction conditions in aqueous alkaline media. Polymer-free transfer to create suspended SLG minimizes contributions to doping related to any support or contaminants, such that changes in work function (WF) relate predominantly to the electrochemical system under study. These WF changes are rationalized in the context of a simple model of electrochemical gating, providing insight into the interplay between electronic and electrochemical doping (through redox of water) of suspended graphene. Further changes in WF are attributable to the reversible functionalization of graphene during the oxygen evolution reaction. Mechanical changes in the suspended graphene in the form of bulging also occur, which are attributed to electro-wetting of graphene induced by charge-carrier doping

Electronic interactions and stability issues at the copper-graphene interface in air and in alkaline solution under electrochemical control

A micro-electrochemical cell is sealed with a polymer-free single-layer graphene (SLG) membrane to monitor the stability of Cu nanoparticles (NPs) attached to SLG, as well as the interfacial electronic interactions between Cu NPs and SLG both in air and in a mildly alkaline aqueous solution under electrochemical control. A combination of techniques, including in-situ Kelvin probe force microscopy (KPFM) and ex-situ electron microscopy, are applied. When Cu NPs are metallic at cathodic potentials, there is a relatively bias-independent offset in the SLG work function due to charge transfer at the Cu-SLG contact. When Cu NPs are oxidized at anodic potentials, on the other hand, the work function of SLG also depends on the applied bias in a quasi-linear fashion due to electrochemical gating, in addition to charge transfer at the CuOx-SLG contact. Furthermore, Cu NPs were found to oxidize and detach from SLG when kept under anodic potentials for a few hours, whereas they remain adhered to SLG at cathodic potentials. This is attributed to water intercalation at the CuO-SLG interface associated with the enhanced hydrophilicity of positively polarized graphene, as supported by the absence of Cu detachment following oxidation by galvanic corrosion in air

Characterization of Eocene flint

Eocene flint 48–56.0 million years old (mya) from the Negev desert (Israel) was characterized using a suite of analytical techniques. High-resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) of the inorganic component showed the texture, morphology, size, and distribution of two silica polymorphs: α–quartz and moganite. While euhedral forms were attributed to α-quartz, moganite crystals were comprised of spherulitic grains. An electron less-dense amorphous material (no scattering under SAED) was found between the siliceous crystallites. Energy dispersive X-rays (EDS) and electron energy loss spectroscopy (EELS) demonstrated that this electron less-dense amorphous material is composed solely of carbon. Low vacuum, low energy backscattered environmental scanning electron microscopy (BSE-eSEM) imaging of flint surfaces showed the presence of micrometer-sized organic inclusions randomly distributed throughout the siliceous matrix. Energy-dispersive X-ray studies (EDS) demonstrated that these organic micro-inclusions were composed of carbon, sulfur, and nitrogen with a C/N ratio attributed to marine sources. These micro-inclusions were not directly associated with hard-shell fossils. BSE-eSEM imaging conditions allowed the identification of entrapped carbon-rich organic material, which is not possible when applying commonly used electron microscopy conditions that require carbon coating and high acceleration voltages, rendering carbon-rich features electron-transparent. Phase contrast-enhanced micro-computed tomography (PC-μCT) showed that these organic micro-inclusions were randomly distributed throughout the siliceous matrix. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), nano-Fourier transform infrared spectroscopy (nano-FTIR), and scanning probe microscopy (SPM) were used to further characterize these organic micro-inclusions. These three in situ analytical techniques with nanometer resolution provided complementary information on the chemical composition and structure of the organic material. Specifically, ToF-SIMS analysis revealed amino acid and hydrocarbon mass spectra fingerprints inside the organic micro-inclusions. While the former were exclusively found in the organic micro-inclusions, the mass spectral fingerprints for hydrocarbons were also found in the siliceous matrix in agreement with the HR-TEM/EDS/EELS results, where pure carbon was found between the siliceous nanocrystals. While ToF-SIMS provides chemical information, it does not provide structural information. Nano-FTIR analysis showed the presence of amide I and II infrared vibrations exclusively on the organic micro-inclusions. The scanning probe microscopy (SPM) techniques Peak Force Quantitative Nanomechanics (PF-QNM) and Contact Resonance Atomic Force Microscopy (CR-AFM) were used to assess the mechanical properties. PF-QNM measurements on the organic micro-inclusions, under dry and liquid conditions, demonstrated that the organic micro-inclusions swell upon hydration and soften, pointing toward the presence of hydrophilic molecules in agreement with nano-FTIR and ToF-SIMS results. CR-AFM allows in situ determination of the mechanical properties of materials with high stiffness at nanometer resolution. This technique, rarely used in a geological context, revealed that the organic micro-inclusions had an unusually high stiffness atypical for modern organic material, which was attributed to molecular cross-linking promoted by diagenesis. This work provided a comprehensive view of the inorganic and organic components of Eocene flint from the Negev desert with implications for paleontology and archaeology. It offers a roadmap of novel complementary techniques that can be used in the exploration of entrapped organic material in flint

20S proteasomes secreted by the malaria parasite promote its growth

Mature red blood cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, secrete within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve human RBCs by remodeling their cytoskeletal network. Furthermore, we identify four degradation targets of the secreted 20S proteasome, the phosphorylated cytoskeletal proteins β-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome secretion mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth