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Summary: Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high‐pressure freezing and freeze‐substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze‐substitution with per‐mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time‐efficient FIB‐SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM‐TEM workflows to comprehensively analyse biological structures. Lay Description: Focused ion beam scanning electron microscopy is becoming a widely used technique for the three‐dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy‐based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub‐cellular and sub‐organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl‐embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures. [ABSTRACT FROM AUTHOR]

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread 1,2 . PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses 3-5 . Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

In Eukaryotes, DNA is wound around the histone octamer forming the basic chromatin unit, the nucleosome. Atomic structures have been obtained from crystallography and single particle cryo-electron microscopy (cryoEM) of identical engineered particles. But native nucleosomes are dynamical entities with diverse DNA sequence and histone content, and little is known about their conformational variability, especially in the cellular context. Using cryoEM and tomography of vitreous sections we analyse native nucleosomes, both in vitro, using purified particles solubilized at physiologically relevant concentrations (25-50%), and in situ, within interphase nuclei. We visualize individual nucleosomes at a level of detail that allows us to measure the distance between the DNA gyres wrapped around. In concentrated solutions, we demonstrate a salt-dependent transition, with a high salt compact conformation resembling the canonical nucleosome and an open low salt one, closer to nuclear nucleosomes. Although further particle characterization and cartography are needed to understand the relationship between this conformational variability and chromatin functional states, this work opens a route to chromatin exploration in situ.

The game of Go has long been viewed as the most challenging of classic games for artificial intelligence owing to its enormous search space and the difficulty of evaluating board positions and moves. Here we introduce a new approach to computer Go that uses 'value networks' to evaluate board positions and 'policy networks' to select moves. These deep neural networks are trained by a novel combination of supervised learning from human expert games, and reinforcement learning from games of self-play. Without any lookahead search, the neural networks play Go at the level of state-of-the-art Monte Carlo tree search programs that simulate thousands of random games of self-play. We also introduce a new search algorithm that combines Monte Carlo simulation with value and policy networks. Using this search algorithm, our program AlphaGo achieved a 99.8% winning rate against other Go programs, and defeated the human European Go champion by 5 games to 0. This is the first time that a computer program has defeated a human professional player in the full-sized game of Go, a feat previously thought to be at least a decade away.

Rarely occurring flavan-3-ol derivatives such as C-glycosides can be generated during food processing, for example, by cocoa production. These astringent taste compounds may also exert interesting behavior toward microbial metabolism, as other C-glycosides have been shown to be quite stable. Oligomeric flavan-3-ols, the procyanidins, bear also a C-C bond between the main moieties and are suspected to resist microbial metabolism for a prolonged time compared to other flavonoids. This paper describes a semisynthetic approach for the generation of flavan-3-ol C-glycosides. Results of incubation experiments studying five flavan-3-ol C-glycosides bearing different sugars, linkage positions, and stereochemistries are presented as well as the behavior of di- and trimeric B-type procyanidins toward intestinal microbiota. Low molecular weight degradation products are considered as well as concentration-time courses of degraded and liberated compounds. All metabolic studies were performed with the well-proven pig cecum model.

Programmed cell death involves the activation of caspase proteases that can mediate the cleavage of vital cytoskeletal proteins. We have recently reported that, in failing cardiac myocytes, caspase-3 activation is associated with a reduction in contractile performance. In this study we used a modified yeast two-hybrid system to screen for caspase-3 interacting proteins of the cardiac cytoskeleton. We identified ventricular essential myosin light chain (vMLC1) as a target for caspase-3. By sequencing and site-directed mutagenesis, a noncanonical cleavage site for caspase-3 was mapped to the C-terminal DFVE(135)G motif. We demonstrated that vMLC1 cleavage in failing myocardium in vivo is associated with a morphological disruption of the organized vMLC1 staining of sarcomeres, and with a reduction in myocyte contractile performance. Adenoviral gene transfer of the caspase inhibitor p35 in vivo prevented caspase-3 activation and vMLC1 cleavage, with positive impact on contractility. These data suggest that direct cleavage of vMLC1 by activated caspase-3 may contribute to depression of myocyte function by altering cross-bridge interaction between myosin and actin molecules. Therefore, activation of apoptotic pathways in the heart may lead to contractile dysfunction before cell death.