Links between memory, synapses, amyloid-beta, tau and microglial changes after tau pathology induction by Alzheimer's brain inoculation in amyloid-beta-plaque bearing mice BackgroundAlzheimers disease (AD) is characterized by the presence of both extracellular amyloid-{beta} (A{beta}) plaques and intracellular tau accumulations. The causative interactions between these lesions, and the subsequent neuroinflammation, synaptic alterations and memory impairment are still debated. Intracerebral infusion of human AD brain extracts in A{beta} bearing mice that do not overexpress pathological tau proteins induces widespread A{beta} and tau pathologies following heterotopic seeding of mouse tau. These models provide a unique opportunity to assess relationships between AD lesions and downstream events.

MethodsHuman AD and control-brain extracts (ADbe and Ctrlbe) were infused in the hippocampus of A{beta} plaque-bearing APPswe/PS1dE9 mice. We evaluated the links between A{beta} plaques, tau aggregates, microgliosis, astrogliosis at the inoculation site and in connected regions (perirhinal/entorhinal cortex), and memory impairment and synaptic density 4 and 8 months post-inoculation.

ResultsADbe-inoculated animals displayed memory deficit and synaptic loss in addition to A{beta} and tau pathological aggregates. A{beta} plaques were detected in both ADbe- and Ctrlbe-inoculated APPswe/PS1dE9 mice, but their load was increased close to the inoculation site in ADbe- inoculated animals. Tau-positive neuropil threads and neurofibrillary tangles occurred next to the inoculation site only in ADbe-inoculated animals and were not detected in Ctrlbe mice. These lesions spread to connected regions notably the perirhinal/entorhinal cortex. Tau-positive neuritic plaques were detected in both ADbe- and Ctrlbe-inoculated animals. Unexpectedly, ADbe inoculation did not increase the number of neuritic plaques close to the inoculation site as compared to Ctrlbe inoculation. ADbe inoculation however increased the area of tau-positive elements within these neuritic plaques. Increased tau pathology as well as lower Iba1-positive microglial load in the hippocampus and perirhinal/entorhinal cortex were correlated with memory impairment and with synaptic loss.

ConclusionsThe integrative analysis of pathological events associated with ADbe inoculation in A{beta}-bearing mice highlighted memory and synaptic loss as well as the induction of tau lesions that spread in the brain. Increased tau lesions and lower microglial load are the two main events correlated with memory impairment and synaptic loss. The study suggests that microglial activity may be protective.

Graphical abstract

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Using Deep Learning to Predict Human Decisions, and Cognitive Models to Explain Deep Learning Models Deep neural networks (DNN) models have the potential to provide new insights in the study of human decision making, due to their high capacity and data-driven design. While these models may be able to go beyond theory-driven models in predicting human behaviour, their opaque nature limits their ability to explain how an operation is carried out. This explainability problem remains unresolved. Here we demonstrate the use of a DNN model as an exploratory tool to identify predictable and consistent human behaviour in value-based decision making beyond the scope of theory-driven models. We then propose using theory-driven models to characterise the operation of the DNN model. We trained a DNN model to predict human decisions in a four-armed bandit task. We found that this model was more accurate than a reinforcement-learning reward-oriented model geared towards choosing the most rewarding option. This disparity in accuracy was more pronounced during times when the expected reward from all options was similar, i.e., no unambiguous good option. To investigate this disparity, we introduced a reward-oblivious model, which was trained to predict human decisions without information about the rewards obtained from each option. This model captured decision-sequence patterns made by participants (e.g., a-b-c-d). In a series of experimental offline simulations of all models we found that the general model was in line with a reward-oriented models predictions when one option was clearly better than the others.

However, when options expected rewards were similar to each other, it was in-line with the reward-oblivious models pattern completion predictions. These results indicate the contribution of predictable but task-irrelevant decision patterns to human decisions, especially when task-relevant choices are not immediately apparent. Importantly, we demonstrate how theory-driven cognitive models can be used to characterise the operation of DNNs, making them a useful explanatory tool in scientific investigation.

Author SummaryDeep neural networks (DNN) models are an extremely useful tool across multiple domains, and specifically for performing tasks that mimic and predict human behaviour. However, due to their opaque nature and high level of complexity, their ability to explain human behaviour is limited. Here we used DNN models to uncover hitherto overlooked aspects of human decision making, i.e., their reliance on predictable patterns for exploration. For this purpose, we trained a DNN model to predict human choices in a decision-making task. We then characterised this data-driven model using explicit, theory-driven cognitive models, in a set of offline experimental simulations. This relationship between explicit and data-driven approaches, where high-capacity models are used to explore beyond the scope of established models and theory-driven models are used to explain and characterise these new grounds, make DNN models a powerful scientific tool.
Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. SERCA function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca2+ uptake were unattainable due to the severe impairments in their ability to transport Ca2+. Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice. Carbon Dot-based Fluorescent Antibody Nanoprobes as Brain Tumour Glioblastoma Diagnostics. The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood, and it relies on fluorescent reporters.

Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne-azide cycloaddition (SPAAC) ligation chemistry. The new class of Ab-CD conjugates developed using this strategy was successfully used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) validating the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems. Fine-scale coexistence between Mediterranean mesocarnivores is mediated by spatial, temporal and trophic resource partitioning O_LIThe partition of the ecological niche can enhance the coexistence of predators due to differences in how they exploit three main resources: food, space, and time, the latter being an axis that often remains unexplored.
C_LIO_LIWe studied niche segregation in a Mediterranean mesocarnivore community composed by Vulpes vulpes, Genetta genetta, Meles meles and Herpestes ichneumon, addressing simultaneously different niche axes: the temporal, trophic and spatial axes.
C_LIO_LIWe assessed temporal segregation between mesopredators and prey and between potential competitors, using camera trap data between 2018-2020 in a Mediterranean landscape in Southern Spain. We deployed camera traps in 35 stations in three sites with varying vegetation cover within Donana National Park. We further examined the spatial overlap in activity centers and trophic preferences between potential competitors using diet information from studies performed in the study area.
C_LIO_LIWe found an overall temporal segregation between trophic generalist species, with species showing higher temporal overlap differing in their trophic preferences and/or showing limited spatial overlap. Furthermore, we observed an overall high overlap between the activity patterns of predators and their major prey in the area (the common genet vs small mammals and the red fox vs European rabbit).
C_LIO_LIOur study suggests that coexistence of the different species that compose the mesocarnivore assemblage in Mediterranean landscapes can be facilitated by subtle differences along the three main niche axes, with temporal segregation being a most pronounced mechanism. Our findings reinforce the idea that the coexistence mechanisms underlying community structure are multidimensional.
C_LI Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.

MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.

ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.

ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Interplay between disordered regions in RNAs and proteins modulates interactions within stress granules and processing bodies Condensation, or liquid-like phase separation, is a phenomenon indispensable for the spatiotemporal regulation of molecules within the cell. Recent studies indicate that the composition and molecular organization of phase-separated organelles such as Stress Granules (SGs) and Processing Bodies (PBs) are highly variable and dynamic. A dense contact network involving both RNAs and proteins controls the formation of SGs and PBs and an intricate molecular architecture, at present poorly understood, guarantees that these assemblies sense and adapt to different stresses and environmental changes. Here, we investigated the physico-chemical properties of SGs and PBs components and studied the architecture of their interaction networks. We found that proteins and RNAs establishing the largest amount of contacts in SGs and PBs have distinct structural properties and intrinsic disorder is enriched in all protein-RNA, protein-protein and RNA-RNA interaction networks. The increase of disorder in proteins is accompanied by an enrichment in single-stranded regions of RNA binding partners. Our results suggest that SGs and PBs quickly assemble and disassemble through fuzzy-like dynamic contacts modulated by unfolded domains of their components.

Research HighlightsO_LIWe systematically studied RNA-RNA, protein-protein and RNA-protein interaction networks in stress granules and processing bodies;
C_LIO_LIRNAs enriched in stress granules and processing bodies are more single-stranded and form a large number of contacts with both proteins and RNAs;
C_LIO_LIProteins in stress granules and processing bodies are less structured and contact larger amounts of single-stranded RNAs.
C_LI Microbial colonization of tannin-rich tropical plants: interplay between degradability, methane production and tannin disappearance in the rumen AO_SCPLOWBSTRACTC_SCPLOWCondensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonization. This study explored the rumen degradation of tropical tannins-rich plants and the relationship between condensed tannins fractions disappearance and microbial communities colonising plant particles. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of dairy cows. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important. Precartilage condensation during limb skeletogenesis occurs by tissue phase separation controlled by a bistable cell-state switch with suppressed oscillatory dynamics The tetrapod limb skeleton is initiated in unpatterned limb bud mesenchyme by the formation of precartilage condensations. Here, based on time-lapse videographic analysis of a forming condensation in a high-density culture of chicken limb bud mesenchyme, we observe a phase transition to a more fluidized state for cells within spatial compacted foci (protocondensations that will progress to condensations), as reflected in their spatial confinement, cell-substratum interaction and speed of motion. Previous work showed that galectin-8 and galectin-1A, two proteins of the galactoside-binding galectin family, are the earliest determinants of this process in the chicken limb bud, and that their interactions in forming skeletogenic patterns of condensations can be interpreted mathematically through a reaction-diffusion-adhesion framework. Based on this framework, we use an ordinary differential equation-based approach to analyze the core switching modality of the galectin reaction network and characterize the states of the network independent of the diffusive and adhesive arms of the patterning mechanism. We identify two steady states where the concentrations of both galectins are respectively, negligible, and very high. An explicit Lyapunov function shows that there are no periodic solutions. For sigmoidal galectin production terms, the model exhibits a bistable switch that arises from a monostable state via saddle-node bifurcation. Our model therefore predicts that the galectin network exists in low and high expression states separated in space or time without any intermediate states. This provides a causal basis for the observed outside vs. inside transition observed in the in vitro video data. We performed a quantitative analysis of the distribution of galectin-1A in cultures of condensing chick limb mesenchymal cells and found that the interior of the protocondensations had concentrations of this protein (compared to the immediate exterior) over and above that expected from its higher cell density, consistent with the models predictions. The galectin-based patterning network is thus suggested, on theoretical grounds, to incorporate a core switch independent of any spatial or temporal dynamics, that drives the chondrogenic cell state transition in limb skeletogenesis. Robustness in regulatory networks of Epithelial Mesenchymal Plasticity as a function of positive and negative feedback loops Epithelial-Mesenchymal plasticity (EMP) is a key arm of cancer metastasis and is observed across many contexts. Cells undergoing EMP can reversibly switch between three classes of phenotypes: Epithelial (E), Mesenchymal (M), and Hybrid E/M. While a large number of multistable regulatory networks have been identified to be driving EMP in various contexts, the exact mechanisms and design principles that enable robustness in driving EMP across contexts are not yet fully understood. Here we investigated dynamic and structural robustness in EMP networks with regards to phenotypic distribution and plasticity. We use two different approaches to simulate these networks: a computationally inexpensive, parameter-independent continuous state space boolean model, and an ODE-based parameter-agnostic framework (RACIPE), both of which yield similar phenotypic distributions. Using perturbations to network topology and by varying network parameters, we show that multistable EMP networks are structurally and dynamically more robust as compared to their randomized counterparts, thereby highlighting their topological hallmarks. These features of robustness are governed by a balance of positive and negative feedback loops embedded in these networks. Using a combination of the number of negative and positive feedback loops weighted by their lengths and sign, we identified a metric that can explain the structural and dynamical robustness of these networks. This metric enabled us to compare networks across multiple sizes, and the network principles thus obtained can be used to identify fragilities in large networks without simulating their dynamics. Our analysis highlights a network topology-based approach to quantify robustness in multistable EMP networks.

Significance StatementEpithelial-Mesenchymal plasticity (EMP) is a key arm of cancer metastasis. Despite extensive intra- and inter-tumor heterogeneity, the characteristics of EMP have been observed to be robust across multiple contexts. We hypothesize that topology of EMP regulatory networks contributes towards this robustness. Here, we measure the robustness of EMP in the form of its phenotypic heterogeneity and multistability and show that EMP networks are more robust to dynamical (change in kinetic parameters) and structural (change in network topology) perturbations as compared to their random network counterparts. Furthermore, we propose a network topology-based metric using the nature and length of feedback loops that explains the observed robustness. Our metric hence serves to quantify robustness in multistable EMP networks without simulating their dynamics. Getting more out of FLAG-Tag co-immunoprecipitation mass spectrometry experiments using FAIMS. Co-immunoprecipitation of proteins coupled to mass spectrometry is critical for the understanding of protein interaction networks. In instances where a suitable antibody is not available, it is common to graft synthetic tags onto target protein sequences and allowing the use of commercially available antibodies for affinity purification. A common approach is through FLAG-Tag co-immunoprecipitation. To allow the selective elution of protein complexes, competitive displacement using a large molar excess of the tag peptides is often carried out. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of these peptides. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device prior to mass spectrometry analysis can be applied to FLAG-Tag co-immunoprecipitation experiment to increase the depth of protein coverage. By excluding these abundant tag peptides, we were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without the need for additional sample handling or altering sample preparation protocols. Juvenile Gouldian Finches (Erythrura gouldiae) form sibling sub-groups during social integration The formation of social relationships in complex groups is critical in shaping patterns of social organization and behavioral development. In many birds, young individuals remain dependent on their parents for extended periods but must abruptly transition to navigating interactions in the wider group after independence. While lack of social relationships during this period is detrimental in the development of later social skills, little is known about the social relationships juveniles form after independence in many bird species. In this study we describe patterns of social interactions in juvenile Gouldian Finches (Erythrura gouldiae) during transitions from family groups into flocks of unfamiliar individuals. A group of 20 juveniles from 4 families were introduced into two flocks. After introductions juveniles showed a gradient of approach rates with most approaches directed towards siblings, followed by juvenile peers, adult females, and lastly adult males. Significant preferences for siblings resulted in the emergence of sibling subgroups within the larger social network. This active self-assortment of siblings suggests that sibling sub-groups are an important bridge linking social connections within the family and the wider group. Such findings suggests that sibling relationships have a critical role in the socialization after independence, as well as structuring the social organization of Gouldian finch flocks. Marine gregarine genomes reveal the breadth of apicomplexan diversity and provide new insights on gliding motility Our current view of the evolutionary history, coding and adaptive capacities of Apicomplexa, protozoan parasites of a wide range of metazoan, is currently strongly biased toward species infecting humans, as data on early diverging apicomplexan lineages infecting invertebrates is extremely limited. Here, we characterized the genome of the marine eugregarine Porospora gigantea, intestinal parasite of Lobsters, remarkable for the macroscopic size of its vegetative feeding forms (trophozoites) and its gliding speed, the fastest so far recorded for Apicomplexa. Two highly syntenic genomes named A and B were assembled. Similar in size (~9 Mb) and coding capacity (~5300 genes), A and B genomes are 10.8% divergent at the nucleotide level, corresponding to 16-38 My in divergent time. Orthogroup analysis across 25 (proto)Apicomplexa species, including Gregarina niphandrodes, showed that A and B are highly divergent from all other known apicomplexan species, revealing an unexpected breadth of diversity. Phylogenetically these two species branch sister to Cephaloidophoroidea, and thus expand the known crustacean gregarine superfamily. The genomes were mined for genes encoding proteins necessary for gliding, a key feature of apicomplexans parasites, currently studied through the molecular model called glideosome. Sequence analysis shows that actin-related proteins and regulatory factors are strongly conserved within apicomplexans. In contrast, the predicted protein sequences of core glideosome proteins and adhesion proteins are highly variable among apicomplexan lineages, especially in gregarines. These results confirm the importance of studying gregarines to widen our biological and evolutionary view of apicomplexan species diversity, and to deepen our understanding of the molecular bases of key functions enabling parasitism, such as the glideosome. Outer hair cell electro-mechanical power conversion Outer hair cells are the cellular motors in the mammalian inner ear responsible for sensitive high-frequency hearing. Motor function requires expression of the protein prestin in the OHC lateral membrane and mechano-electrical transduction in the apical hair bundle. In the present report, electrical power consumption and mechanical power output are determined using previously published voltage clamp data from isolated OHCs and membrane patches. Results reveal that power output by the prestin-motor complex peaks at a best frequency much higher than implied by the low-pass character of nonlinear capacitance, and much higher than the whole-cell resistive-capacitive corner frequency. High frequency power output is enabled by a -90{degrees} shift in the phase of prestin-dependent electrical charge displacement, manifested electrically as emergence of imaginary-valued nonlinear capacitance. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. spatialGE: Quantification and visualization of the tumor microenvironment heterogeneity using spatial transcriptomics SummarySpatially-resolved transcriptomics promises to increase our understanding of the tumor microenvironment and improve cancer prognosis and therapies. Nonetheless, analytical methods to explore associations between the spatial heterogeneity of the tumor and clinical data are not available. Hence, we have developed spatialGE, a software that provides visualizations and quantification of the tumor microenvironment heterogeneity through gene expression surfaces, spatial heterogeneity statistics (SThet) that can be compared against clinical information, spot-level cell deconvolution, and spatially-informed clustering (STclust), all using a new data object to store data and resulting analyses simultaneously.

Availability and implementationThe R package and tutorial/vignette are available at https://github.com/FridleyLab/spatialGE. A script to reproduce the analyses in this manuscript is available in Supplementary information.

ContactFridley.Lab@Moffitt.org or Brooke.Fridley@Moffitt.org

Supplementary informationAvailable at Bioinformatics online.



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C_FIG Host factors drive the within-host competition between highly and low pathogenic H5N8 avian influenza viruses Highly pathogenic avian influenza viruses (HPAIV) emerge from low pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse-genetics engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. By contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variants interactions in viral evolution. Mechanical loading of cranial joints minimizes the craniofacial phenotype in Crouzon syndrome Children with syndromic forms of craniosynostosis undergo a plethora of surgical interventions to resolve the clinical features caused by the premature fusion of cranial sutures. While surgical correction is reliable, the need for repeated rounds of invasive treatment puts a heavy burden on the child and their family.

This study explores a non-surgical alternative using mechanical loading of the cranial joints to prevent or delay craniofacial phenotypes associated with Crouzon syndrome.

We treated Crouzon syndrome mice before the onset of craniosynostosis by cyclical mechanical loading of cranial joints using a custom designed set-up.

Cranial loading applied to the frontal bone partially restores normal skull morphology, significantly reducing the typical brachycephalic appearance. This is underpinned by the delayed closure of the coronal suture and of the intersphenoidal synchondrosis.

This study provides a novel treatment alternative for syndromic craniosynostosis which has the potential to be an important step towards replacing, reducing or refining the surgical treatment of all craniosynostosis patients. An orthologous gene coevolution network provides insight into eukaryotic cellular and genomic structure and function Orthologous gene coevolution--which refers to gene pairs whose evolutionary rates covary across speciation events--is often observed among functionally related genes. We present a comprehensive gene coevolution network inferred from the examination of nearly three million orthologous gene pairs from 332 budding yeast species spanning [~]400 million years of eukaryotic evolution. Modules within the network provide insight into cellular and genomic structure and function, such as genes functioning in distinct cellular compartments and DNA replication. Examination of the phenotypic impact of network perturbation across 14 environmental conditions using deletion mutant data from the bakers yeast Saccharomyces cerevisiae suggests that fitness in diverse environments is impacted by orthologous gene neighborhood and connectivity. By mapping the network onto the chromosomes of S. cerevisiae and the opportunistic human pathogen Candida albicans, which diverged [~]235 million years ago, we discovered that coevolving orthologous genes are not clustered in either species; rather, they are most often located on different chromosomes or far apart on the same chromosome. The budding yeast coevolution network captures the hierarchy of eukaryotic cellular structure and function, provides a roadmap for genotype-to-phenotype discovery, and portrays the genome as an extensively linked ensemble of genes. Tunable Cellular Localization and Extensive Cytoskeleton-Interplay of Reflectins Reflectin proteins are nature copolymers consist of repeated canonical domains, locating in a biophotonic system called Bragg lamellae and manipulating the dynamic structural coloration of iridocytes. Their biological functions are intriguing, but the underlying mechanism is not fully understood. In this study, reflectin A1, A2, B1 and C were found to present distinguished cyto-/nucleoplasmic localization preferences. Comparable intracellular localization can be reproduced by truncated reflectin variants, suggesting a conceivable evolutionary order among reflectin proteins. Secondly, the size-dependent access of reflectin variants into nucleus demonstrate a potential model of how reflectins get into Bragg lamellae. Thirdly, RfA1 was found to extensively interact with cytoskeleton, including its binding to actin and microtubule minus-end-directed movement. This implies cytoskeleton system plays fundamental role during the organization and transportation of reflectin proteins. Findings presented here recommend reflectins as programmable biomaterials which can be used to decipher their evolution processes, to delineate their biological mechanism, and to achieve tunable intracellular targeting as editable tags. Plant inositol-phosphate-glycans and a fucosylated xyloglucan oligosaccharide are accumulated upon Arabidopsis thaliana/ Botrytis cinerea infection In mammals, insulin is involved in controlling blood glucose levels and its role in modulating immunity is being more and more documented. This hormone promotes the release of inositolphosphate glycans (IPG) which act as mediators. In plants, one IG has already been identified in plant culture cells (Smith and Fry, 1999; Smith et al., 1999) but, to our knowledge, no IPG have been yet identified. Here, we discovered 7 IPG that are accumulated upon Arabidopsis thaliana-Botrytis cinerea interaction, concomitantly with oligogalacturonides and a fucosylated xyloglucan oligosaccharide. Further structural characterization showed that they come from the hydrolysis of polar heads of Serie A to H glycosylinositol phosphorylceramides presumably via a phospholipase C activity. Taken together with the emerging role of insulin as immune regulator, these results question the role of IPG as damage associated molecular pattern both in animal and plant kingdoms. Temporal and Spatial Dynamics of Hepatic Stellate Cell Activation in Intrahepatic Cholangiocarcinoma Intrahepatic cholangiocarcinoma (iCCA) is an aggressive type of primary liver cancer characterized by its highly desmoplastic stroma. Compared to the ample knowledge on cancer-associated fibroblast residing within iCCA tumor mass, little is known about the fibrotic response from the tumor-surrounding liver and its role in iCCA development. In this study, we find a significant accumulation of peritumoral myofibroblasts (pMFs) in both patient iCCA and tumors from an iCCA orthotopic allograft mouse model. Using tumor and liver spheroid coculture we show that iCCA-liver interaction induces rapid pMF accumulation at the interface. We find pMFs placed around iCCA spheroids exert a strong suppressive effect on tumor cells growth, in contrast to the pro-proliferative effect of MFs mixed within tumor spheroids. However, prolonged iCCA-pMF interaction elicits tumor cell dissemination in vitro. We find an upregulation of Vcam1 in tumor cells in the early phase of iCCA-pMF interaction both in vitro and in vivo which is downregulated when tumor cells disseminate. Blocking Vcam1 activity in iCCA allograft mouse models slows primary tumor growth but lead to increased tumor metastasis. Our data suggest that pMFs are beyond simple pro- or anti-tumorigenic in iCCA, with the ability to suppress tumor growth but elicit tumor cell dissemination. Changes to the mtDNA copy number during yeast culture growth We show that the mtDNA copy number in growing cultures of the yeast Saccharomyces cerevisiae increases by a factor of up to 4, being lowest ([~]10 per haploid genome) and stable during rapid fermentative growth, and highest at the end of the respiratory phase. When yeast are grown on glucose, the onset of the mtDNA copy number increase coincides with the early stages of the diauxic shift, and the increase continues through respiration. A lesser yet still substantial copy number increase occurs when yeast are grown on a nonfermentable carbon source, i.e. when there is no diauxic shift. The mtDNA copy number increase during and for some time after the diauxic shift is not driven by an increase in cell size. The copy number increase occurs in both haploid and diploid strains, but is markedly attenuated in a diploid wild isolate that is a ready sporulator. Strain-to-strain differences in mtDNA copy number are least apparent in fermentation and most apparent in late respiration or stationary phase. While changes in mitochondrial morphology and function were previously known to accompany changes in physiological state, it had not been previously shown that the mtDNA copy number changes substantially over time in a clonal growing culture. The mtDNA copy number in yeast is therefore a highly dynamic phenotype. Embryonic exposure to cannabidiol disrupts active neural circuits, an effect increased by Δ9-tetrahydrocannabinol and involving CB1R and CB2R in zebrafish In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development.