ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Growth hormone remodels the 3D-structure of the mitochondria of inflammatory macrophages and promotes metabolic reprogramming.

Introduction: Macrophages are a heterogeneous population of innate immune cells that support tissue homeostasis through their involvement in tissue development and repair, and pathogen defense. Emerging data reveal that metabolism may control macrophage polarization and function and, conversely, phenotypic polarization may drive metabolic reprogramming. Methods: Here we use biochemical analysis, correlative cryogenic fluorescence microscopy and cryo-focused ion-beam scanning electron microscopy. Results: We demonstrate that growth hormone (GH) reprograms inflammatory GM-CSF-primed monocyte-derived macrophages (GM-MØ) by functioning as a metabolic modulator. We found that exogenous treatment of GM-MØ with recombinant human GH reduced glycolysis and lactate production to levels similar to those found in anti-inflammatory M-MØ. Moreover, GH treatment of GM-MØ augmented mitochondrial volume and altered mitochondrial dynamics, including the remodeling of the inner membrane to increase the density of cristae. Conclusions: Our data demonstrate that GH likely serves a modulatory role in the metabolism of inflammatory macrophages and suggest that metabolic reprogramming of macrophages should be considered as a new target to intervene in inflammatory diseases.

Interplay between septins and ubiquitin-mediated xenophagy during Shigella entrapment.

Septins are cytoskeletal proteins implicated in numerous cellular processes including cytokinesis and morphogenesis. In the case of infection by Shigella flexneri, septins assemble into cage-like structures that entrap cytosolic bacteria targeted by autophagy. The interplay between septin cage entrapment and bacterial autophagy is poorly understood. We used a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study septin cage entrapment of Shigella in its near-native state. Septin cages could be identified as X-ray dense structures, indicating they contain host cell proteins and lipids consistent with their autophagy links. Airyscan confocal microscopy of Shigella-septin cages showed that septins and lysine 63 (K63)-linked ubiquitin chains are present in separate bacterial microdomains, suggesting they are recruited separately. Finally, Cryo-SXT and live-cell imaging revealed an interaction between septins and microtubule-associated protein light chain 3B (LC3B)-positive membranes during autophagy of Shigella. Collectively our data present a new model for how septin-caged Shigella are targeted to autophagy.

Septins and K63 ubiquitin chains are present in separate bacterial microdomains during autophagy of entrapped Shigella.

During host cell invasion, Shigella escapes to the cytosol and polymerizes actin for cell-to-cell spread. To restrict cell-to-cell spread, host cells employ cell-autonomous immune responses including antibacterial autophagy and septin cage entrapment. How septins interact with the autophagy process to target Shigella for destruction is poorly understood. Here, we employed a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study Shigella septin cage entrapment in its near-native state. Quantitative cryo-SXT showed that Shigella fragments mitochondria and enabled visualization of X-ray-dense structures (∼30 nm resolution) surrounding Shigella entrapped in septin cages. Using Airyscan confocal microscopy, we observed lysine 63 (K63)-linked ubiquitin chains decorating septin-cage-entrapped Shigella. Remarkably, septins and K63 chains are present in separate bacterial microdomains, indicating they are recruited separately during antibacterial autophagy. Cryo-SXT and live-cell imaging revealed an interaction between septins and LC3B-positive membranes during autophagy of Shigella. Together, these findings demonstrate how septin-caged Shigella are targeted for autophagy and provide fundamental insights into autophagy-cytoskeleton interactions.

Photobio-electrocatalytic production of H2 using fluorine-doped tin oxide (FTO) electrodes covered with a NiO-In2S3 p-n junction and NiFeSe hydrogenase.

Clean energy vectors are needed towards a fossil fuel-free society, diminishing both greenhouse effect and pollution. Electrochemical water splitting is a clean route to obtain green hydrogen, the cleanest fuel; although efficient electrocatalysts are required to avoid high overpotentials in this process. The combination of inorganic semiconductors with biocatalysts for photoelectrochemical H2 production is an alternative approach to overcome this challenge. N-type semiconductors can be coupled to a co-catalyst for H2 production in the presence of a sacrificial electron donor in solution, but the replacement of the latter with an electrode is a challenge. In this work we attach a NiFeSe-hydrogenase with high activity for H2 production with the n-type semiconductor indium sulfide, which upon visible irradiation is able to transfer its excited electrons to the enzyme. In order to enhance the transfer of the generated holes towards the electrode for their replenishment, we have explored the inclusion of a p-type material, NiO, to induce a p-n junction for H2 production in a photoelectrochemical biocatalytic system in absence of sacrificial reagents.

Efficient Production of Solar Hydrogen Peroxide Using Piezoelectric Polarization and Photoinduced Charge Transfer of Nanopiezoelectrics Sensitized by Carbon Quantum Dots.

Piezoelectric semiconductors have emerged as redox catalysts, and challenges include effective conversion of mechanical energy to piezoelectric polarization and achieving high catalytic activity. The catalytic activity can be enhanced by simultaneous irradiation of ultrasound and light, but the existing piezoelectric semiconductors have trouble absorbing visible light. A piezoelectric catalyst is designed and tested for the generation of hydrogen peroxide (H2 O2 ). It is based on Nb-doped tetragonal BaTiO3 (BaTiO3 :Nb) and is sensitized by carbon quantum dots (CDs). The photosensitizer injects electrons into the conduction band of the semiconductor, while the piezoelectric polarization directed electrons to the semiconductor surface, allowing for a high-rate generation of H2 O2 . The piezoelectric polarization field restricts the recombination of photoinduced electron-hole pairs. A production rate of 1360 µmol gcatalyst -1  h-1  of H2 O2  is achieved under visible light and ultrasound co-irradiation. Individual piezo- and photocatalysis yielded lower production rates. Furthermore, the CDs enhance the piezocatalytic activity of the BaTiO3 :Nb. It is noted that moderating the piezoelectricity of BaTiO3 :Nb via microstructure modulation influences the piezophotocatalytic activity. This work shows a new methodology for synthesizing H2 O2  by using visible light and mechanical energy.

Promotion of Ru or Ni on Alumina Catalysts with a Basic Metal for CO2 Hydrogenation: Effect of the Type of Metal (Na, K, Ba).

Ru and Ni on alumina catalysts have been promoted with a 10 wt% of alkali metal (K or Na) or alkaline earth metal (Ba) and tested in CO2 methanation. For the catalyst consisting of Ni and Ba, the variation of Ba loading while keeping Ni loading constant was studied. The promotion in terms of enhanced CH4 yield was found only for the addition of barium to 15 wt% Ni/Al2O3. In contrast, K and Na addition increased the selectivity to CO while decreasing conversion. For the Ru-based catalyst series, no enhancement in conversion or CH4 yield was attained by any of the alkaline metals. CO2 temperature-programed desorption (CO2-TPD) revealed that the amount of chemisorbed CO2 increased significantly after the addition of the base metal. The reactivity of COx ad-species for each catalyst was assessed by temperature-programed surface reaction (TPSR). The characterization revealed that the performance in the Sabatier reaction was a result of the interplay between the amount of chemisorbed CO2 and the reactivity of the COx ad-species, which was maximized for the (10%Ba)15%Ni/Al2O3 catalyst.

Multiphoton imaging of melanoma 3D models with plasmonic nanocapsules.

We report the synthesis of plasmonic nanocapsules and the cellular responses they induce in 3D melanoma models for their perspective use as a photothermal therapeutic agent. The wall of the nanocapsules is composed of polyelectrolytes. The inner part is functionalized with discrete gold nanoislands. The cavity of the nanocapsules contains a fluorescent payload to show their ability for loading a cargo. The nanocapsules exhibit simultaneous two-photon luminescent, fluorescent properties and X-ray contrasting ability. The average fluorescence lifetime (τ) of the nanocapsules measured with FLIM (0.3 ns) is maintained regardless of the intracellular environment, thus proving their abilities for bioimaging of models such as 3D spheroids with a complex architecture. Their multimodal imaging properties are exploited for the first time to study tumorspheres cellular responses exposed to the nanocapsules. Specifically, we studied cellular uptake, toxicity, intracellular fate, generation of reactive oxygen species, and effect on the levels of hypoxia by using multi-photon and confocal laser scanning microscopy. Because of the high X-ray attenuation and atomic number of the gold nanostructure, we imaged the nanocapsule-cell interactions without processing the sample. We confirmed maintenance of the nanocapsules' geometry in the intracellular milieu with no impairment of the cellular ultrastructure. Furthermore, we observed the lack of cellular toxicity and no alteration in oxygen or reactive oxygen species levels. These results in 3D melanoma models contribute to the development of these nanocapsules for their exploitation in future applications as agents for imaging-guided photothermal therapy. STATEMENT OF SIGNIFICANCE: The novelty of the work is that our plasmonic nanocapsules are multimodal. They are responsive to X-ray and to multiphoton and single-photon excitation. This allowed us to study their interaction with 2D and 3D cellular structures and specifically to obtain information on tumor cell parameters such as hypoxia, reactive oxygen species, and toxicity. These nanocapsules will be further validated as imaging-guided photothermal probes.

High Performance Generation of H2 O2 under Piezophototronic Effect with Multi-Layer In2 S3 Nanosheets Modified by Spherical ZnS and BaTiO3 Nanopiezoelectrics.

Manipulating the separation and transportation of photoexcited charge carriers in photoresponsive semiconductors via the piezoelectric polarization effect is an emerging strategy in the field of artificial photosynthesis. However, existing semiconductor photocatalysts, both with a wide range absorption for visible light and superior piezoelectricity are very scarce, leading to a low reactivity of photocatalysis. Here, a multi-layer In2 S3 nanosheet modified with spherical ZnS and BaTiO3 nanopiezoelectrics (ZnS/In2 S3 /BTO) is reported, generating approximately 378 µm of H2 O2 in 100 min (and the concentration is still increasing) under co-irradiation of visible light and ultrasound (piezophotocatalysis) in ethanol-water solution; this concentration is higher compared with two phases piezoelectric heterostructures (i.e., ZnS/BTO, In2 S3 /BTO, and ZnS/In2 S3 ) and pure compounds (i.e., ZnS, In2 S3 , and BTO), and also higher than that of independent piezo- (≈254 µm) and photocatalysis (≈120 µm). Moreover, the concentration of H2 O2 generated on ZnS/In2 S3 /BTO can be as high as approximately 1160 µm in 5 h of piezophotoreaction after experiencing six cycles of visible light concurrent with ultrasound irradiation. The enhancement of H2 O2 yield on ZnS/In2 S3 /BTO in piezophotocatalysis can be attributed to the piezopotential-induced internal electric polarization field promoting the separation of photoexcited charge carriers, thus boosting the rate of surface photoreaction.

Structural analysis of receptors and actin polarity in platelet protrusions.

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbß3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Computing with DFT Band Offsets at Semiconductor Interfaces: A Comparison of Two Methods.

Two DFT-based methods using hybrid functionals and plane-averaged profiles of the Hartree potential (individual slabs versus vacuum and alternating slabs of both materials), which are frequently used to predict or estimate the offset between bands at interfaces between two semiconductors, are analyzed in the present work. These methods are compared using several very different semiconductor pairs, and the conclusions about the advantages of each method are discussed. Overall, the alternating slabs method is recommended in those cases where epitaxial mismatch does not represent a significant problem.

Calcite as a Precursor of Hydroxyapatite in the Early Biomineralization of Differentiating Human Bone-Marrow Mesenchymal Stem Cells.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce.

Image Processing in Cryo-Electron Microscopy of Single Particles: The Power of Combining Methods.

Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.

Multimodal Imaging and Soft X-Ray Tomography of Fluorescent Nanodiamonds in Cancer Cells.

Multimodal imaging promises to revolutionize the understanding of biological processes across scales in space and time by combining the strengths of multiple imaging techniques. Fluorescent nanodiamonds (FNDs) are biocompatible, chemically inert, provide high contrast in light- and electron-based microscopy, and are versatile optical quantum sensors. Here it is demonstrated that FNDs also provide high absorption contrast in nanoscale 3D soft X-ray tomograms with a resolution of 28 nm in all dimensions. Confocal fluorescence, atomic force, and scanning electron microscopy images of FNDs inside and on the surface of PC3 cancer cells with sub-micrometer precision are correlated. FNDs are found inside ≈1 µm sized vesicles present in the cytoplasm, providing direct evidence of the active uptake of bare FNDs by cancer cells. Imaging artefacts are quantified and separated from changes in cell morphology caused by sample preparation. These results demonstrate the utility of FNDs in multimodal imaging, contribute to the understanding of the fate of FNDs in cells, and open up new possibilities for biological imaging and sensing across the nano- and microscale.

Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage.

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.

Atomic-Scale Model and Electronic Structure of Cu2O/CH3NH3PbI3 Interfaces in Perovskite Solar Cells.

Cuprous oxide has been conceived as a potential alternative to traditional organic hole-transport layers in hybrid halide perovskite-based solar cells. Device simulations predict record efficiencies using this semiconductor, but experimental results do not yet show this trend. More detailed knowledge about the Cu2O/perovskite interface is mandatory to improve the photoconversion efficiency. Using density functional theory calculations, here, we study the interfaces of CH3NH3PbI3 with Cu2O to assess their influence on device performance. Several atomistic models of these interfaces are provided for the first time, considering different compositions of the interface atomic planes. The interface electronic properties are discussed on the basis of the optimal theoretical situation, but in connection with the experimental realizations and device simulations. It is shown that the formation of vacancies in the Cu2O terminating planes is essential to eliminate dangling bonds and trap states. The four interface models that fulfill this condition present a band alignment favorable for photovoltaic conversion. Energy of adhesion and charge transfer across the interfaces are also studied. The termination of CH3NH3PbI3 in PbI2 atomic planes seems optimal to maximize the photoconversion efficiency.

Multiparametric analysis of the effectiveness of cisplatin on cutaneous squamous carcinoma cells using two different types of adjuvants.

The objective of this study was to regulate the cytotoxicity of cisplatin (cisPt) minimizing its adverse effects. For this purpose, the lowest cisPt concentration needed to obtain a significant positive response in cutaneous squamous cell carcinoma (cSCC) was explored. Two adjuvant agents as gold nanoparticles (AuNP) and chelating tricine were tested as enhancers in cisPt treatment. Effectiveness of all treatments was assessed by means of biochemical techniques, which offer quantitative data, as well as two microscopy-based techniques that provided qualitative cell imaging. The present work confirms the effectiveness of free cisplatin at very low concentrations. In order to enhance its effectiveness while the side effects were probably diminished, cisPt 3.5 µM was administered with AuNP 2.5 mM, showing an effectiveness practically equal to that observed with free cisPt. However, the second treatment investigated, based on cisPt 3.5 µM combined with tricine 50 mM, enhanced drug effectiveness, increasing the percentage of cells dying by apoptosis. This treatment was even better in terms of cell damage than free cisPt at 15 µM. Images obtained by TEM and cryo-SXT confirmed these results, since a notable number of apoptotic bodies were detected when cisPt was combined with tricine. Thus, tricine was clearly a better adjuvant for cisPt treatments.

Unambiguous Intracellular Localization and Quantification of a Potent Iridium Anticancer Compound by Correlative 3D Cryo X-Ray Imaging.

The iridium half-sandwich complex [Ir(η5 :κ1 -C5 Me4 CH2 py)(2-phenylpyridine)]PF6 is highly cytotoxic: 15-250× more potent than clinically used cisplatin in several cancer cell lines. We have developed a correlative 3D cryo X-ray imaging approach to specifically localize and quantify iridium within the whole hydrated cell at nanometer resolution. By means of cryo soft X-ray tomography (cryo-SXT), which provides the cellular ultrastructure at 50 nm resolution, and cryo hard X-ray fluorescence tomography (cryo-XRF), which provides the elemental sensitivity with a 70 nm step size, we have located the iridium anticancer agent exclusively in the mitochondria. Our methodology provides unique information on the intracellular fate of the metallodrug, without chemical fixation, labeling, or mechanical manipulation of the cells. This cryo-3D correlative imaging method can be applied to a number of biochemical processes for specific elemental localization within the native cellular landscape.

Structures of T7 bacteriophage portal and tail suggest a viral DNA retention and ejection mechanism.

Double-stranded DNA bacteriophages package their genome at high pressure inside a procapsid through the portal, an oligomeric ring protein located at a unique capsid vertex. Once the DNA has been packaged, the tail components assemble on the portal to render the mature infective virion. The tail tightly seals the ejection conduit until infection, when its interaction with the host membrane triggers the opening of the channel and the viral genome is delivered to the host cell. Using high-resolution cryo-electron microscopy and X-ray crystallography, here we describe various structures of the T7 bacteriophage portal and fiber-less tail complex, which suggest a possible mechanism for DNA retention and ejection: a portal closed conformation temporarily retains the genome before the tail is assembled, whereas an open portal is found in the tail. Moreover, a fold including a seven-bladed ß-propeller domain is described for the nozzle tail protein.

Encapsulation, Visualization and Expression of Genes with Biomimetically Mineralized Zeolitic Imidazolate Framework-8 (ZIF-8).

Recent work in biomolecule-metal-organic framework (MOF) composites has proven to be an effective strategy for the protection of proteins. However, for other biomacromolecules such as nucleic acids, the encapsulation into nano MOFs and the related characterizations are in their infancy. Herein, encapsulation of a complete gene-set in zeolitic imidazolate framework-8 (ZIF-8) MOFs and cellular expression of the gene delivered by the nano MOF composites are reported. Using a green fluorescent protein (GFP) plasmid (plGFP) as a proof-of-concept genetic macromolecule, successful transfection of mammalian cancer cells with plGFP for up to 4 days is shown. Cell transfection assays and soft X-ray cryo-tomography (cryo-SXT) demonstrate the feasibility of DNA@MOF biocomposites as intracellular gene delivery vehicles. Expression occurs over relatively prolonged time points where the cargo nucleic acid is released gradually in order to maintain sustained expression.