Miracle-dz/UniFAR-FTUs huggingface dataset:
Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

The structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method in recent years. Many results of cryo-EM SPA are based on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment. Here, we used a multi-purpose TEM with a side entry cryo-holder at our facility to evaluate the potential of high-resolution SPA. We report a 3 Å resolution map of apoferritin with local resolution extending to 2.6 Å. The map clearly showed two positions of an aromatic side chain. We also verified the optimal imaging conditions depending on different electron microscope and camera combinations. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.
Examining the relationship between social cognition and neural synchrony during movies in children with and without autism

Children who have been diagnosed with autism spectrum disorder (ASD) often show a marked deficit in measures of social cognition. In autistic adults, measures of social cognition have been shown to relate to differences in brain synchronization (as measured by fMRI) when individuals are processing naturalistic stimuli, such as movies. However, whether children with social impairments, with or without a diagnosis of ASD, differ in their neural responses to movies has not yet been investigated. In the current study, neural synchrony, measured using fMRI, was examined in three groups of children aged 7 to 12, who differed with respect to scores on a measure of autistic traits associated with social impairment and whether or not they had been diagnosed with ASD. While watching the movie ‘Despicable Me’, those diagnosed with ASD had significantly less neural synchrony in areas that have been previously shown to be associated with social cognition (e.g. areas related to ‘theory of mind’), and plot following (e.g. the lateral prefrontal cortex), than those who did not have an ASD diagnosis. In contrast, two groups who differed in their degree of social impairment, but did not have a diagnosis of ASD, showed no significant differences in neural synchrony across the whole brain. These results shed some light on how autistic traits may contribute to an individual’s conscious experience of the world, and how, for children with ASD, that experience may differ markedly from that of those without ASD.
Motif analysis in co-expression networks reveals regulatory elements in plants: The peach as a model

Identification of functional regulatory elements encoded in plant genomes is a fundamental need to understand gene regulation. While much attention has been given to model species as Arabidopsis thaliana, little is known about regulatory motifs in other plant genera. Here, we describe an accurate bottom-up approach using the online workbench RSAT::Plants for a versatile ab-initio motif discovery taking Prunus persica as a model. These predictions rely on the construction of a co-expression network to generate modules with similar expression trends and assess the effect of increasing upstream region length on the sensitivity of motif discovery. Applying two discovery algorithms, 18 out of 45 modules were found to be enriched in motifs typical of well-known transcription factor families (bHLH, bZip, BZR, CAMTA, DOF, E2FE, AP2-ERF, Myb-like, NAC, TCP, WRKY) and a novel motif. Our results indicate that small number of input sequences and short promoter length are preferential to minimize the amount of uninformative signals in peach. The spatial distribution of TF binding sites revealed an unbalanced distribution where motifs tend to lie around the transcriptional start site region. The reliability of this approach was also benchmarked in Arabidopsis thaliana, where it recovered the expected motifs from promoters of genes containing ChIPseq peaks. Overall, this paper presents a glimpse of the peach regulatory components at genome scale and provides a general protocol that can be applied to many other species. Additionally, a RSAT Docker container was released to facilitate similar analyses on other species or to reproduce our results. One sentence summary Motifs prediction depends on the promoter size. A proximal promoter region defined as an interval of -500 bp to +200 bp seems to be the adequate stretch to predict de novo regulatory motifs in peach
A unified approach for characterizing static/dynamic connectivity frequency profiles using filter banks

Static and dynamic functional network connectivity (FNC) are typically studied separately, which makes us unable to see the full spectrum of connectivity in each analysis. Here, we propose an approach called filter-banked connectivity (FBC) to estimate connectivity while preserving its full frequency range and subsequently examine both static and dynamic connectivity in one unified approach. First, we demonstrate that FBC can estimate connectivity across multiple frequencies missed by a sliding-window approach. Next, we use FBC to estimate FNC in a resting-state fMRI dataset including schizophrenia patients (SZ) and typical controls (TC). The FBC results are clustered into different network states. Some states showed weak low-frequency strength and as such were not captured in the window-based approach. Additionally, we found that SZs tend to spend more time in states exhibiting higher frequencies compared with TCs who spent more time in lower frequency states. Finally, we show that FBC enables us to analyze static and dynamic connectivity in a unified way. In summary, FBC offers a novel way to unify static and dynamic connectivity analyses and can provide additional information about the frequency profile of connectivity patterns.
Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos

Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and indicate that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.
Should virus capsids assemble perfectly? Theory and observation of defects

Although published structural models of viral capsids generally exhibit a high degree of regularity or symmetry, structural defects might be expected for several reasons: the finite-temperature, fluctuating environment in which capsids assemble, the maturation process many capsids undergo, and the requirement of some capsids for disassembly prior to genome delivery. Different lines of evidence also suggest the presence of defects: the observation of defective structures in computer simulations, and the presence of imperfect and off-pathway capsids in single-particle cryoEM studies. To begin quantifying the conditions under which defects might be expected, we extend prior work by developing a simple equilibrium theory for capsids based on a lattice model allowing for both ideal and defective sites. Both analytical and numerical calculations with the model show there is a fairly sharp threshold value for the difference in effective pair-binding affinities between ideally oriented and mixed ideal/defective pairs: below this capsid-specific threshold, there is an appreciable population of defective capsids. Even when defective sites are not allowed, there is generally some population of vacancies. Our theoretical analysis is bolstered by quantitation of single particles in cryoEM micrographs suggesting that ≳ 15% of particles may be defective. Beyond furthering our understanding of structural heterogeneity in virus capsids, the findings suggest a non-traditional strategy for assembly inhibition by enhancing ‘off-lattice’ interactions among capsid proteins via adaptor molecules. Significance Statement Both computer simulations of icosahedral viral capsid assembly and cryoelectron (cryoEM) microscopy images of assembled capsids show that capsids sometimes assemble into defective structures of lesser symmetry. We found that about 15% of cryoEM images of single hepatitis B virus capsids show defective capsids. To begin to understand capsid defects on a quantitative basis, we developed a model with sites that can either be vacant, ideally occupied, or mis-occupied. Defects were generally found to occur, with a frequency dependent on the interaction strengths of ideally occupied sites and misoccupied sites. Our results suggest that enhancing incorrect interactions between capsid proteins may be a way to diminish correct assembly.
Integrated metabolomics and proteomics of symptomatic and early pre-symptomatic states of colitis

Two murine models for colitis were used to study multi-level changes and derive molecular signatures of colitis onset and development. By combining metabolomics data on tissues and fecal extracts with proteomics data on tissues, we provide a comprehensive picture of the metabolic profile of acute and chronic states of the disease, and most importantly, of two early pre-symptomatic states. We show that, increased anaerobic glycolysis, accompanied by altered TCA cycle and oxidative phosphorylation, associates with inflammation-induced hypoxia taking place in colon tissues. We also demonstrate significant changes in the metabolomic profiles of fecal extracts in different colitis states, most likely associated with the dysbiosis characteristic of colitis, as well as the dysregulated tissue metabolism. Most remarkably, strong and distinctive tissue and fecal metabolomic signatures can be detected before onset of symptoms. These results highlight the diagnostic potential of global metabolomics for inflammatory diseases.
A molecular map of lymph node blood vascular endothelium at single cell resolution

Blood vascular endothelial cells (BECs) control the immune response by regulating immune cell recruitment, metabolite exchange and blood flow in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain poorly understood. Here we profile transcriptomes of BEC from mouse peripheral lymph nodes and map key phenotypes to the vasculature. Our analysis identifies multiple novel subsets including a venous population whose gene signature predicts an unexpectedly selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by 2 photon videomicroscopy. We define five phenotypes of capillary lining BEC including a capillary resident regenerative population (CRP) that displays stem cell and migratory gene signatures and contributes to homeostatic BEC turnover and to vascular neogenesis after immunization. Trajectory analyses reveal retention of developmental programs along a progression of cellular phenotypes from CRP to mature venous and arterial BEC subsets. Overall, our single cell atlas provides a molecular blueprint of the lymph node blood vasculature and defines subset specialization for immune cell recruitment and vascular homeostasis.
Molecular Scale Spatio-Chemical Control of the Activating-Inhibitory Signal Integration in NK Cells

The role of the spatial juxtaposition between activating and inhibitory receptors in cytotoxic lymphocytes has been strongly debated in the context of the inhibition of immune signaling. The challenge in addressing this problem was so far a lack of experimental tools which can simultaneously manipulate different signaling molecules. Here, we circumvent this challenge by introducing a nanoengineered multifunctional cell niche, in which activating and inhibitory ligands are positioned with molecular-scale variability and control, and applied it to elucidate the role of the spatial juxtaposition between ligands for NKG2D and KIR2DL1 – activating and inhibitory receptors in Natural Killer (NK) cells – in KIR2DL1-mediated inhibition of NKG2D signaling. We realized the niche by a nanopatterning of nanodots of different metals with molecular scale registry in one lithographic step, followed by a novel ternary functionalization of the fabricated bi-metallic pattern and its background to with three distinct biochemical moieties. We found, that within the probed range, the 40 nm gap between the activating and inhibitory ligands provided an optimal inhibition condition. Supported by theoretical modeling and simulations we interpret these findings as a consequence of the size and conformational flexibility of the ligands in their spatial interaction. Our findings provide an important insight onto the spatial mechanism of the inhibitory immune checkpoints, whose understanding is both fundamentally important, and essential for the rational design of future immunotherapies. Furthermore, our approach is highly versatile and paves the way to numerous complex molecular platforms aimed at revealing molecular mechanisms through which receptors integrate their signals.
A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

Membrane fusion, an essential process in all eukaryotes, is driven by SNARE proteins. Ykt6 is an essential SNARE that plays critical roles throughout the secretory, endocytic, and autophagy pathways. Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. Through genetic, pharmacologic, and structural modeling approaches in mammalian cells, we found that phosphorylation regulates Ykt6 conversion from a closed to an open state. The phosphorylation site we identified is highly conserved in evolution and is regulated by the Ca2+-dependent phosphatase, calcineurin. We found that phosphorylation is a key determinant for intracellular localization of Ykt6 and its function in macroautophagy. Our studies reveal a novel mechanism by which Ykt6 conformation and activity is regulated by Ca2+ signaling with implications in Parkinson’s Disease in which Ykt6 has been shown to play a role.
Belowground changes to community structure alter methane-cycling dynamics in Amazonia

Amazonian rainforest is undergoing increasing rates of deforestation, driven primarily by cattle pasture expansion. Forest-to-pasture conversion has been associated with changes to ecosystem processes, including substantial increases in soil methane (CH4) emission. The drivers of this change in CH4 flux are not well understood. To address this knowledge gap, we measured soil CH4 flux, environmental conditions, and belowground microbial community attributes across a land use change gradient (old growth primary forest, cattle pasture, and secondary forest regrowth) in two Amazon Basin regions. Primary forest soils exhibited CH4 uptake at modest rates, while pasture soils exhibited CH4 emission at high but variable rates. Secondary forest soils exhibited low rates of CH4 uptake, suggesting that forest regrowth following pasture abandonment could reverse the CH4 sink-to-source trend. While few environmental variables were significantly associated with CH4 flux, we identified numerous microbial community attributes in the surface soil that explained substantial variation in CH4 flux with land use change. Among the strongest predictors were the relative abundance and diversity of methanogens, which both increased in pasture relative to forests. We further identified individual taxa that were associated with CH4 fluxes and which collectively explained ~50% of flux variance. These taxa included methanogens and methanotrophs, as well as taxa that may indirectly influence CH4 flux through acetate production, iron reduction, and nitrogen transformations. Each land type had a unique subset of taxa associated with CH4 fluxes, suggesting that land use change alters CH4 cycling through shifts in microbial community composition. Taken together, our results suggest that changes in CH4 flux from agricultural conversion could be driven by microbial responses to land use change in the surface soil, with both direct and indirect effects on CH4 cycling. This demonstrates the central role of microorganisms in mediating ecosystem responses to land use change in the Amazon Basin.
The effect of acute sleep deprivation on skeletal muscle protein synthesis and the hormonal environment

Chronic sleep loss is a potent catabolic stressor, increasing the risk of metabolic dysfunction and loss of muscle mass and function. To provide mechanistic insight into these clinical outcomes, we sought to determine if acute sleep deprivation blunts skeletal muscle protein synthesis and promotes a catabolic environment. Healthy young adults (N=13; 7 male, 6 female) were subjected to one night of total sleep deprivation (DEP) and normal sleep (CON) in a randomized cross-over design. Anabolic and catabolic hormonal profiles, skeletal muscle fractional synthesis rate and markers of muscle protein degradation were assessed across the following day. Acute sleep deprivation reduced muscle protein synthesis by 18% (CON: 0.072 ± 0.015 vs. DEP: 0.059 ± 0.014 %•h-1, p=0.040). In addition, it increased plasma cortisol by 21% (p=0.030) and decreased plasma testosterone, but not IGF-1, by 22% (p=0.029). A single night of total sleep deprivation is sufficient to induce anabolic resistance and a pro-catabolic environment. These acute changes may represent mechanistic precursors driving the metabolic dysfunction and body composition changes associated with chronic sleep deprivation.
Neurons in the ventral striatopallidal complex modulate lateral hypothalamic orexin/hypocretin neuron activity: Implications for reward-seeking

Reward-seeking involves the engagement and computation of multiple physiological and motivational parameters. The lateral hypothalamus (LH) is a necessary node in the circuits that control food-seeking and motivation. One group of cells that plays an important yet incompletely understood role in these processes are the orexin/hypocretin (OX/HT) neurons. OX/HT cells are located exclusively within the LH and are implicated in feeding, arousal, and reward-seeking behavior. Importantly, the role of OX/HT neurons in consummatory versus reward-seeking actions is not fully defined, nor are the circuits that control the activity of these neurons under different behavioral states. Here we show that OX/HT neurons respond in real time to food presentation and that this response is modulated by differences in metabolic state. We observed increased OX/HT neuron activity on approach to food, but this activity trended towards pre-approach levels by the start of the consummatory phase. Next, we studied ventrostriatopallidal (VSP) inputs to the OX/HT neurons. Using optogenetics and cell type-specific electrophysiology, we found that ventral pallidum inputs onto OX/HT neurons exert strong inhibitory (and weak excitatory) effects whereas the lateral nucleus accumbens shell provides weaker direct inhibitory connections with OX/HT neurons. These findings reveal that the activity of OX/HT neurons is strongly modulated by metabolic and hedonic state. Further, OX/HT neurons is primarily associated with food approach and that the effect of VSP-terminal output is to suppress OX/HT activity.
The physical form of microbial ligands bypasses the need for dendritic cell migration to stimulate adaptive immunity

A central paradigm of immunology is that the innate immune system first detects infectious agents in peripheral tissues, shortly after a pathogen has breached an epithelial barrier. This detection event is mediated by pattern recognition receptors in phagocytes, which then migrate to draining lymph nodes (dLNs), where information of a microbial encounter is conveyed to T and B lymphocytes to generate adaptive immunity. Through the study of fungal moieties, we present data that challenge this model. We found that soluble fungal polysaccharides are immunosilent in the periphery, but become potent immunogens in the dLN. These ligands completely bypass the need of phagocyte migration and, instead, directly activate an immune response that is most similar to those that typify viral infections. These data establish a class of microbial products that violate a central tenet of the immunological lexicon and illustrate that the physical form (not just the chemical structure) impacts innate and adaptive immunity.
Solanaceae specialized metabolism in a non-model plant: trichome acylinositol biosynthesis

Plants make hundreds of thousands of biologically active specialized metabolites varying widely in structure, biosynthesis and the processes that they influence. An increasing number of these compounds are documented to protect plants from harmful insects, pathogens, or herbivores, or mediate interactions with beneficial organisms including pollinators and nitrogen fixing microbes. Acylsugars – one class of protective compounds – are made in glandular trichomes of plants across the Solanaceae family. While most described acylsugars are acylsucroses, published examples also include acylsugars with hexose cores. The South American fruit crop Solanum quitoense (Naranjilla) produces acylsugars that contain a myo-inositol core. We identified an enzyme that acetylates triacylinositols, a function homologous to the last step in the Solanum lycopersicum acylsucrose biosynthetic pathway. Our analysis reveals parallels between S. lycopersicum acylsucrose and S. quitoense acylinositol biosynthesis, suggesting a common evolutionary origin. Material availability The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Robert L. Last (lastr@msu.edu). One sentence summary Evidence that the final step in Solanum quitoense acylinositol biosynthesis evolved from an acylsucrose acetyltransferase enzyme.