bluuebunny/crossref_metadata_2025 huggingface dataset:
High-throughput ligand profile characterization in novel biosensor cell lines expressing seven heterologous olfactory receptors for the detection of volatile plant biomarkers
Authors: Zboray Katalin , Toth Adam Viktor , Miskolczi Timea Dora , Casanova Emilio , Mike Arpad , Sagi Laszlo , Lukacs Peter
Published: April 2023

Agriculturally important crop plants emit a multitude of volatile organic compounds (VOCs), which are excellent indicators of their health status and/or their interaction with pathogens and pests. Here, we present the generation of a novel cellular biosensor panel for the recognition of fungal pathogen-related VOCs we had identified in the field as well as during controlled inoculations of several crop plants. The panel consists of seven stable HEK293 cell lines each expressing a functional Drosophila olfactory receptor as a biosensing element and a genetically encoded fluorescent calcium indicator protein. For high-throughput measurement of odorant binding by the cells, fluorescence response was detected in an automated 384-well microplate reader upon the injection of tester VOCs. Biosensor cell lines were characterized for their reference ligand binding in more detail, then a set of 66 VOCs was profiled on all cell lines over a concentration range of three orders of magnitude (1 μM to 100 μM). Forty-six VOCs (70%) evoked a response in at least one biosensor cell line and certain VOCs could activate the cell lines already from the nanomolar (ppb) concentration. Interaction profiles mapped in this study will support biosensor development for agricultural applications, but the olfactory receptor proteins may also be purified from these cell lines at sufficient yields for further processing including structure determination or coupling with artificial sensor devices.
Designing host-associated microbiomes using the consumer/resource model
Authors: Plata Germán , Srinivasan Karthik , Krishnamurthy Madan , Herron Lukas , Dixit Purushottam
Published: April 2023

A key step towards rational microbiome engineering isin silicosampling of realistic microbial communities that correspond to desired host phenotypes, and vice versa. This remains challenging due to a lack of generative models that simultaneously capture compositions of host-associated microbiomes and host phenotypes. To that end, we present a generative model based on the mechanistic consumer/resource (C/R) framework. In the model, variation in microbial ecosystem composition arises due to differences in the availability ofeffective resources(inferred latent variables) while species’ resource preferences remain conserved. The same latent variables are used to model phenotypic states of hosts.In silicomicrobiomes generated by our model accurately reproduce universal and dataset-specific statistics of bacterial communities. The model allows us to address three salient questions in host-associated microbial ecologies: (1) which host phenotypes maximally constrain the composition of the host-associated microbiomes? (2) how context-specific are phenotype/microbiome associations, and (3) what are plausible microbiome compositions that correspond to desired host phenotypes? Our approach aids the analysis and design of microbial communities associated with host phenotypes of interest.
Thermoprotection by a cell membrane-localized metacaspase in a green alga
Authors: Zou Yong , Sabljić Igor , Horbach Natalia , Dauphinee Adrian N. , Åsman Anna , Temino Lucia Sancho , Drag Marcin , Stael Simon , Poreba Marcin , Ståhlberg Jerry , Bozhkov Peter V.
Published: April 2023

Caspases are restricted to animals, while other organisms, including plants possess metacaspases (MCAs), a more ancient and broader class of structurally-related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis expressing nine MCAs with partly redundant activities. In contrast to streptophytes, most chlorophytes contain only one or two hitherto uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigate CrMCA-II, a single type II MCA from a model chlorophyteChlamydomonas reinhardtii. Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes bothin vitroandin vivo. Furthermore, activation of CrMCA-IIin vivocorrelates with the dimerization. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion ofCrMCA-IIby CRISPR/Cas9 compromises thermotolerance leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restores thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we link the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.
The Shank/ProSAP N-terminal (SPN) domain of Shank3 regulates targeting to postsynaptic sites and postsynaptic signalling
Authors: Woike Daniel , Tibbe Debora , Nia Fatemeh Hassani , Martens Victoria , Wang Emily , Barsukov Igor , Kreienkamp Hans-Jürgen
Published: April 2023

Members of the Shank family of postsynaptic scaffold proteins (Shank1-3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, αCaMKII and cytoskeletal proteins. Here we analyse the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive αCaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on αCaMKII activity at postsynaptic sites in micein vivo. The relevance of the SPN-Ank intramolecular interaction was further analysed in primary cultured neurons; here we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of dendritic spines. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signalling, and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.
Neoepitope-specific vaccination of a patient with diffuse midline glioma targeting H3K27M induces polyclonal B and T cell responses across diverse HLA alleles
Authors: Boschert Tamara , Kromer Kristina , Lerner Taga , Lindner Katharina , Haltenhof Gordon , Tan Chin Leng , Jähne Kristine , Poschke Isabel , Bunse Lukas , Grassl Niklas , Mildenberger Iris , Sahm Katharina , Platten Michael , Lindner John M , Green Edward W
Published: April 2023

H3K27M, a driver mutation with T- and B-cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient who was treated with an H3K27M peptide vaccine and subsequently entered complete remission. The vaccine robustly expanded class II HLA-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their CDR3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ, and -DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.
Temperature and day length drive local adaptation in the Patagonian foundation tree speciesNothofagus pumilio
Authors: Sekely Jill , Marchelli Paula , Arana Verónica , Dauphin Benjamin , Mattera María Gabriela , Pastorino Mario , Scotti Ivan , Soliani Carolina , Heer Katrin , Opgenoorth Lars
Published: April 2023

Climate change alters relationships among environmental conditions and thus has the potential to change the selection pressures acting on adaptive gene variants. Using a landscape genomic approach, we show that the southern beech speciesNothofagus pumiliohas notable genetic adaptations to climate along its 2000-kilometer-long range in the Andes. We screened 47,336 SNP loci in 1,632 contigs and found that high-latitude sampling sites have lower genetic diversity, likely due to greater impact of glacial oscillations at high latitudes. Using four genome scan methods, we identified 457 outlier SNPs that are either strongly differentiated among subpopulations or associated with environmental covariates related to temperature, day length, and precipitation. Temperature and day length parameters were associated with notably more outliers than precipitation (n = 133, 113, and 61 outliers, respectively), and almost half of all annotated outliers were related to stress response (n=38, 21%) or catabolism-metabolism (n=43, 24%). Our findings suggest thatNothofagus pumiliois an ideal Andean model of genetic adaptation to climate change because it is locally adapted to extant climate conditions, and shifting patterns among environmental parameters may be detrimental to its future survival and adaptation potential.
Identification of transcription factor co-binding patterns with non-negative matrix factorization
Authors: Rauluseviciute Ieva , Launay Timothée , Barzaghi Guido , Nikumbh Sarvesh , Lenhard Boris , Krebs Arnaud Regis , Castro-Mondragon Jaime A. , Mathelier Anthony
Published: April 2023

Transcription factor (TF) binding to DNA is critical to transcription regulation. Although the binding properties of numerous individual TFs are well-documented, a more detailed comprehension of how TFs interact cooperatively with DNA, forming either complex or co-binding to the same region, is required. Indeed, the combinatorial binding of TFs is essential to cell differentiation, development, and response to external stimuli. We present COBIND, a novel method based on non-negative matrix factorization (NMF) to identify TF co-binding patterns automatically. COBIND applies NMF to one-hot encoded regions flanking known TF binding sites (TFBSs) to pinpoint enriched DNA patterns at fixed distances. We applied COBIND to 8,293 TFBS datasets from UniBind for 404 TFs in seven species. The method uncovered already established co-binding patterns (e.g.,between POU5F1 and SOX2 or SOX17) and new co-binding configurations not yet reported in the literature and inferred through motif similarity and protein-protein interaction knowledge. Our extensive analyses across species revealed that 84% of the studied TFs share a co-binding motif with other TFs from the same structural family. The co-binding patterns captured by COBIND are likely functionally relevant as they harbor higher evolutionarily conservation than isolated TFBSs. Open chromatin data from matching human cell lines further supported the co-binding predictions. Finally, we used single-molecule footprinting data from mouse embryonic stem cells to confirm that the co-binding events captured by COBIND were likely occurring on the same DNA molecules.
Self-supervision advances morphological profiling by unlocking powerful image representations
Authors: Kim Vladislav , Adaloglou Nikolaos , Osterland Marc , Morelli Flavio M. , Halawa Marah , König Tim , Gnutt David , Zapata Paula A. Marin
Published: April 2023

Cell Painting is an image-based assay that offers valuable insights into drug mechanisms of action and off-target effects. However, traditional feature extraction tools such as CellProfiler are computationally intensive and require frequent parameter adjustments. Inspired by recent advances in AI, we trained self-supervised learning (SSL) models DINO, MAE, and SimCLR on subsets of the JUMP-CP dataset to obtain powerful image representations for Cell Painting. We assessed the reproducibility and biological relevance of SSL features and uncovered the critical factors influencing model performance, such as training set composition and domain-specific normalization techniques. Our best model (DINO) surpassed CellProfiler in drug target and gene family classification, significantly reducing computational time and costs. All SSL models showed remarkable generalizability without fine-tuning, outperforming CellProfiler on an unseen dataset of genetic perturbations. Our study demonstrates the effectiveness of SSL methods for morphological profiling, suggesting promising research directions for improving the analysis of related image modalities.
Non-destructive spatial mapping of vegetation plots: a re-introduction of the pantograph
Authors: Martyn Trace E. , Mayfield Margaret M.
Published: April 2023

Non-destructive spatial mapping of herbaceous plants is often not possible with many modern imaging techniques, especially in systems with highly structured, dense herbaceous canopies. For this purpose we suggest using a modern version of the classic pantograph, a simple instrument that allows precisely scaled drawings. The pantograph version we describe here was specifically designed for small-scale herbaceous vegetation mapping.

Specifically, our pantograph design is useful for rapidly collecting accurate, spatially explicit data at the scale of 0.1-2 m2and includes a paired drawing board for easy use in field conditions.

We tested the design and technique on 100 annual plant plots that ranged in total density and in plant stature. Based on this mapping trial, we present guidelines for effective manual mapping and map digitization.

A pantograph is a useful and inexpensive to make tool for non-destructively spatially mapping individual herbaceous plants in the field. Here, we present instructions for the design and fabrication of our modern pantograph, board, and pencil attachment designed specifically for researchers wanting to include small-scale spatial context in their research.
AbNatiV: VQ-VAE-based assessment of antibody and nanobody nativeness for hit selection, humanisation, and engineering
Authors: Ramon Aubin , Ali Montader , Atkinson Misha , Saturnino Alessio , Didi Kieran , Visentin Cristina , Ricagno Stefano , Xu Xing , Greenig Matthew , Sormanni Pietro
Published: April 2023

Monoclonal antibodies have emerged as key therapeutics, and nanobodies are rapidly gaining momentum following the approval of the first nanobody drug in 2019. Nonetheless, the development of these biologics as therapeutics remains a challenge. Despite the availability of established in vitro directed evolution technologies that are relatively fast and cheap to deploy, the gold standard for generating therapeutic antibodies remains discovery from animal immunization or patients. Immune-system derived antibodies tend to have favourable properties in vivo, including long half-life, low reactivity with self-antigens, and low toxicity. Here, we present AbNatiV, a deep-learning tool for assessing the nativeness of antibodies and nanobodies, i.e., their likelihood of belonging to the distribution of immune-system derived human antibodies or camelid nanobodies. AbNatiV is a multi-purpose tool that accurately predicts the nativeness of Fv sequences from any source, including synthetic libraries and computational design. It provides an interpretable score that predicts the likelihood of immunogenicity, and a residue-level profile that can guide the engineering of antibodies and nanobodies indistinguishable from immune-system-derived ones. We further introduce an automated humanisation pipeline, which we applied to two nanobodies. Wet-lab experiments show that AbNatiV-humanized nanobodies retain binding and stability at par or better than their wild type, unlike nanobodies humanised relying on conventional structural and residue-frequency analysis. We make AbNatiV available as downloadable software and as a webserver.
Microbes as part of ancestral neuronal circuits: Bacterial produced signals affect neurons controlling eating behavior inHydra
Authors: Giez Christoph , Pinkle Denis , Giencke Yan , Wittlieb Jörg , Herbst Eva , Spratte Tobias , Lachnit Tim , Klimovich Alexander , Selhuber-Unkel Christine , Bosch Thomas
Published: April 2023

Although recent studies indicate the impact of microbes on the central nervous systems and behavior, it remains unclear how the relationship between the functionality of the nervous system, behavior and the microbiota arise. We studied the eating behavior of Hydra, a host that has a simple nervous system and a low-complexity microbiota. To identify the neuronal subpopulations involved, we used a subpopulation specific cell ablation system and calcium imaging. The role of the microbiota was uncovered by reducing the diversity of the natural microbiota. Here, we demonstrate that different neuronal subpopulations are functioning together to control the eating behavior. The microbiota participates in control of the eating behavior since germ-free or mono-colonized animals have drastic difficulties in mouth opening. This was restored by adding a full complement of the microbiota. In summary, we provide a mechanistic explanation of how the eating behavior is controlled inHydraand how microbes can affect the neuronal circuit.

Highlights

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Multiple neuronal modules and their networks control complex behavior in an animal lacking a central nervous system.

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Its associated microbes participate in these neuronal circuits and influence the eating behavior.

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Disorganization of the microbiota negatively impacts this eating behavior.

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Glutamate participates in an evolutionary ancient interkingdom language.
Suppression of autoimmune arthritis and neuroinflammation via an amino acid-conjugated butyrate prodrug with enhanced oral bioavailability
Authors: Cao Shijie , Budina Erica , Raczy Michal M. , Solanki Ani , Nguyen Mindy , Beckman Taryn N. , Reda Joseph W. , Hultgren Kevin , Ang Phillip , Slezak Anna J. , Hesser Lauren A. , Alpar Aaron T. , Refvik Kirsten C. , Shores Lucas S. , Pillai Ishita , Wallace Rachel P. , Dhar Arjun , Watkins Elyse A. , Hubbell Jeffrey A.
Published: April 2023

Butyrate, a metabolite produced by commensal bacteria, has been intensively studied for its immunomodulatory effects on various immune cells, including T regulatory cells, macrophages, and dendritic cells. Butyrate’s development as a drug has been limited by its poor oral bioavailability due to its rapid metabolism in the gut, its low potency and thus high dosing, and its foul smell and taste. By simply esterifying butyrate to serine (O-butyryl-L-serine, SerBut), a design based on the concept of utilizing amino acid transporters to escape the gut and enhance systemic uptake thus increasing bioavailability, we developed an odorless and tasteless compound for oral administration. In the collagen antibody-induced arthritis (CAIA) and experimental autoimmune encephalomyelitis (EAE) murine models of rheumatoid arthritis and multiple sclerosis, we demonstrated that SerBut significantly ameliorated disease severity, modulated key immune cell populations both systemically and in disease-associated tissues, and reduced inflammatory responses without compromising global immune response to vaccination. Our findings highlight SerBut as a promising next-generation therapeutic agent for autoimmune and inflammatory diseases.
Environmental determinants of suitable habitat and the prediction of a southern shift in the future distribution of spiny lobsters, genusJasus
Authors: Tepker Jason H. E. , Strugnell Jan M. , Silva Catarina N. S.
Published: April 2023

Climate change is altering environmental conditions which is affecting species habitats globally. As a result, many species are shifting their habitat ranges poleward to ensure that they remain within their optimal thermal range. On average, marine species have shifted their ranges poleward by approximately 72km to track their optimal thermal conditions compared to 17km for terrestrial species. These range shifts are pushing some species out of their currently fished areas. This will require nations and fishery companies to predict the most likely areas their target species could move to and obtain permits to fish in those new areas. Spiny lobsters (genusJasus) are likely to shift their ranges poleward as they are distributed within a relatively tight latitudinal band, but there is limited information about the extent of any potential range shifts. The aims of this study were to identify the environmental variables that characterise the current habitat locations of lobsters within the genusJasus, and to predict their potential distribution by modelling future suitable habitat under the RCP45, RCP60, and RCP85 climate scenarios using MaxEnt. There were 16 environmental variables used for modelling suitable habitat for the present (2000-2014), while only four environmental variables were available for modelling in two future time periods (2040-2050 and 2090-2100). There was a predicted overall southern shift in suitable habitat locations for all species. The most important environmental variable identified for species distributed along continental shelves (J. edwardsiiandJ. lalandii) was benthic temperature. Benthic nutrients (silicate, nitrate, and phosphate) were the most important variables for species distributed around islands and on seamounts (J. paulensis, J. frontalis,andJ. tristani). Approximately 90% ofJasuslobsters’ present range contained highly suitable habitat locations. The percent of highly suitable locations under the RCP45 and RCP60 scenarios were higher than the present percentages for each species, while under the RCP85 scenario, there was a decrease of highly suitable habitat for most species in 2040-2050 period, while for the 2090- 2100 period, there was an increase in the percent of highly suitable habitats. This study provides evidence thatJasuspopulations might become more abundant in the southern extents of their current range as they track their optimum habitat conditions.
Phylogenetic variations in a novel family of hyperstable apple snail egg proteins: insights into structural stability and functional trends
Authors: Pasquevich M. Y. , Dreon M. S. , Diupotex-Chong M. E. , Heras H.
Published: April 2023

The relationship between protein stability and function evolution has not been explored in proteins from natural sources. Here, we investigate the phylogenetic differences of Perivitellin-1 (PV1) a novel family of hyperstable egg carotenoproteins crucial to the reproductive success ofPomaceasnails, as they have evolved clade-specific protective functions.

We studiedP. patulaPV1 (PpaPV1) from Flagellata clade eggs, the most basal ofPomaceaand compared it with PV1s orthologs from derived clades. PpaPV1 stands as the most stable, with longer unfolding half-life, resistance to detergent unfolding, and therefore higher kinetic stability than PV1s from derived clades. In fact, PpaPV1 is among the most hyperstable proteins described in nature. In addition, its spectral characteristics providing a pale egg coloration, mild lectin activity and glycan specificity are narrower than derived clades.

Our results provide evidence indicating large structural and functional changes along the evolution of the genus.

Notably, the lectin binding of PpaPV1 is less pronounced, and its glycan specificity is narrower compared to PV1s in the sister Bridgesii clade. Our findings underscore the phylogenetic disparities in terms of structural and kinetic stability, as well as defensive traits like a potent lectin activity affecting the gut morphology of potential predators within the Bridgesii clade or a conspicuous, likely warning coloration, within the Canaliculata clade.

This work provides a comprehensive comparison of the structural attributes, stability profiles, and functional roles of apple snail egg PV1s from multiple species within a phylogenetic context. Furthermore, it proposes an evolutionary hypothesis suggesting a trade-off between structural stability and the functional aspects of apple snail’s major egg defense protein.
Atomic Models of All Major Trans-Envelope Complexes Involved in Lipid Trafficking inEscherichia ColiConstructed Using a Combination of AlphaFold2, AF2Complex, and Membrane Morphing Simulations
Authors: McDonnell Robert T. , Patel Nikhil , Wehrspan Zachary J. , Elcock Adrian H.
Published: April 2023

In Gram-negative bacteria, several trans-envelope complexes (TECs) have been identified that span the periplasmic space in order to facilitate lipid transport between the inner- and outer- membranes. While partial or near-complete structures of some of these TECs have been solved by conventional experimental techniques, most remain incomplete. Here we describe how a combination of computational approaches, constrained by experimental data, can be used to build complete atomic models for four TECs implicated in lipid transport inEscherichia coli. We use DeepMind’s protein structure prediction algorithm, AlphaFold2, and a variant of it designed to predict protein complexes, AF2Complex, to predict the oligomeric states of key components of TECs and their likely interfaces with other components. After obtaining initial models of the complete TECs by superimposing predicted structures of subcomplexes, we use the membrane orientation prediction algorithm OPM to predict the likely orientations of the inner- and outer- membrane components in each TEC. Since, in all cases, the predicted membrane orientations in these initial models are tilted relative to each other, we devise a novel molecular mechanics-based strategy that we call “membrane morphing” that adjusts each TEC model until the two membranes are properly aligned with each other and separated by a distance consistent with estimates of the periplasmic width inE. coli. The study highlights the potential power of combining computational methods, operating within limits set by both experimental data and by cell physiology, for producing useable atomic structures of very large protein complexes.
Rational development of a small-molecule activator of CK1γ2 that decreases C99 and beta-amyloid levels
Authors: Bustos Victor Hugo , Sunkari Yashoda Krishna , Sinha Anjana , Pulina Maria , Bispo Ashley , Hopkins Maya , Lam Alison , Kriegsman Sydney F. , Mui Emily , Chang Emily , Jedlicki Ana , Rosenthal Hannah , Flajolet Marc , Greengard Paul , Sinha Subhash C.
Published: April 2023

Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder characterized by the accumulation of beta-amyloid (Aβ), C99, and Tau in vulnerable areas of the brain. Despite extensive research, current strategies to lower Aβ levels have shown limited efficacy in slowing the cognitive decline associated with AD. Recent findings suggest that C99 may also play a crucial role in the pathogenesis of AD.

Our laboratory has discovered that CK1γ2 phosphorylates Presenilin 1 at the γ-secretase complex, leading to decreased C99 and Aβ levels. Thus, CK1γ2 activation appears as a promising therapeutic target to lower both C99 and Aβ levels.

In this study, we demonstrate that CK1γ2 is inhibited by intramolecular autophosphorylation and describe a high-throughput screen designed to identify inhibitors of CK1γ2 autophosphorylation. We hypothesize that these inhibitors could lead to CK1γ2 activation and increased PS1-Ser367 phosphorylation, ultimately reducing C99 and Aβ levels.

Using cultured cells, we investigated the impact of these compounds on C99 and Aβ concentrations and confirmed that CK1γ2 activation effectively reduces their levels. Our results provide proof of concept that CK1γ2 is an attractive therapeutic target for AD.

Future studies should focus on the identification of specific compounds that can inhibit CK1γ2 autophosphorylation and evaluate their efficacy in preclinical models of AD. These studies will pave the way for the development of novel therapeutics for the treatment of AD.
Neural Signatures of Evidence Accumulation Encode Subjective Perceptual Confidence
Authors: Dou Wei , Martinez Arango Lleymi J. , Castaneda Olenka Graham , Arellano Leopoldo , Mcintyre Emily , Yballa Claire , Samaha Jason
Published: April 2023

Confidence is an adaptive computation when environmental feedback is absent, yet there is little consensus regarding how perceptual confidence is computed in the brain. Difficulty arises because confidence correlates with other factors such as accuracy, response time (RT), or evidence quality. We investigated whether neural signatures of evidence accumulation during a perceptual choice predict subjective confidence independently of these factors. Using motion stimuli, a central-parietal EEG component (CPP) behaves as an accumulating decision variable that predicts evidence quality, RT, accuracy, and confidence (Experiment 1). Psychophysically varying confidence while holding accuracy constant (Experiment 2), the CPP still predicts confidence. Statistically controlling for RT, accuracy, and evidence quality (Experiment 3), the CPP still explains unique variance in confidence. The results indicate that evidence accumulation, indexed by the CPP, is tightly linked to the subjective perceptual experience of sensory information. Independent of other factors, pre-decision neural signatures of evidence accumulation encode subjective confidence.
Prefrontal projections modulate recurrent circuitry in insular cortex to support short-term memory
Authors: Yao Jian , Hou Ruiqing , Fan Hongmei , Hou Jincan , Cheng Qi , Li Chengyu T.
Published: April 2023

Short-term memory (STM) maintains information during a short delay period. How long-range and local connections interact to support STM encoding remains elusive. Here we tackled the problem focusing on long-range projections from medial prefrontal cortex (mPFC) to anterior agranular insular cortex (aAIC) in head-fixed mice performing an olfactory delayed-response task. Optogenetic and electrophysiological experiments revealed behavioral importance of the two regions in encoding STM information. Spike-correlogram analysis revealed strong local and cross-region functional coupling (FC) between memory neurons encoding the same information. Optogenetic suppression of mPFC-aAIC projections during the delay period reduced behavioral performance, the proportion of memory neurons and memory-specific FC within aAIC, whereas optogenetic excitation enhanced all of them. MPFC-to-aAIC projections also bidirectionally modulated efficacy of STM-information transfer, measured by contribution of FC spiking pairs to coding ability of following neurons. Thus, prefrontal projections modulate functional connectivity and coding ability of insular neurons to support short-term memory.
Systematic Pan-cancer Functional Inference and Validation of Hyper, Hypo and Neomorphic Mutations
Authors: Tagore Somnath , Tsang Samuel , Mills Gordon B. , Califano Andrea
Published: April 2023

While the functional effects of many recurrent cancer mutations have been characterized, the TCGA repository comprises more than 10M non-recurrent events, whose function is unknown. We propose that the context specific activity of transcription factor (TF) proteins—as measured by expression of their transcriptional targets—provides a sensitive and accurate reporter assay to assess the functional role of oncoprotein mutations. Analysis of differentially active TFs in samples harboring mutations of unknown significance—compared to established gain (GOF/hypermorph) or loss (LOF/hypomorph) of function—helped functionally characterize 577,866 individual mutational events across TCGA cohorts, including identification of mutations that are either neomorphic (gain of novel function) or phenocopy other mutations (mutational mimicry). Validation using mutation knock-in assays confirmed 15 out of 15 predicted gain and loss of function mutations and 15 of 20 predicted neomorphic mutations. This could help determine targeted therapy in patients with mutations of unknown significance in established oncoproteins.
Epigenetic fidelity in complex biological systems and implications for ageing
Authors: Duffield Thomas , Csuka Laura , Akalan Arda , Vega Magdaleno Gustavo , Senez Ludovic , Palmer Daniel , de Magalhães João Pedro
Published: April 2023

The study of age is plagued by a lack of delineation between the causes and effects within the ageing phenotype. This has made it difficult to fully explain the biological ageing process from first principles with a single definition. Lacking a clear description of the underlying root cause of biological age confounds clarity in this critical field. In this paper, we demonstrate that the epigenetic system has a built-in, unavoidable fidelity limitation and consequently demonstrate that there is a distinct class of DNA methylation loci that increases in variance in a manner tightly correlated with chronological age. We demonstrate the existence of epigenetic ‘activation functions’ and that topological features beyond these activation functions represent deregulation. We show that the measurement of epigenetic fidelity is an accurate predictor of cross-species age and present a deep-learning model that predicts chronological age exclusively from knowledge of variance. We find that the classes of epigenetic loci in which variation correlates with chronological age control genes that regulate transcription and suggest that the inevitable consequence of this is a feedback cycle of system-wide deregulation causing a progressive collapse into the phenotype of age. This paper represents a novel theory of biological systemic ageing with arguments as to why, how and when epigenetic ageing is inevitable.
iPSC-derived healthy human astrocytes selectively load miRNAs targeting neuronal genes into extracellular vesicles
Authors: Gordillo-Sampedro Sara , Antounians Lina , Wei Wei , Mufteev Marat , Lendemeijer Bas , Kushner Steven A. , M.S. de Vrij Femke , Zani Augusto , Ellis James
Published: April 2023

Astrocytes are in constant communication with neurons during the establishment and maturation of functional networks in the developing brain. Astrocytes release extracellular vesicles (EVs) containing microRNA (miRNA) cargo that regulates transcript stability in recipient cells. Astrocyte released factors are thought to be involved in neurodevelopmental disorders. Healthy astrocytes partially rescue Rett Syndrome (RTT) neuron function. EVs isolated from stem cell progeny also correct aspects of RTT. EVs cross the blood-brain barrier (BBB) and their cargo is found in peripheral blood which may allow non-invasive detection of EV cargo as biomarkers produced by healthy astrocytes. Here we characterize miRNA cargo and sequence motifs in healthy human astrocyte derived EVs (ADEVs). First, human induced Pluripotent Stem Cells (iPSC) were differentiated into Neural Progenitor Cells (NPCs) and subsequently into astrocytes using a rapid differentiation protocol. iPSC derived astrocytes expressed specific markers, displayed intracellular calcium transients and secreted EVs. miRNAs were identified by RNA-Seq on astrocytes and ADEVs and target gene pathway analysis detected brain related terms. The miRNA profile was consistent with astrocyte identity, and included approximately 80 miRNAs found in astrocytes that were relatively depleted in ADEVs suggestive of passive loading. About 120 miRNAs were relatively enriched in ADEVs and motif analysis discovered binding sites for RNA binding proteins FUS, SRSF7 and CELF5. miRNA-483-5p was the most significantly enriched in ADEVs. This miRNA regulates MECP2 expression in neurons and has been found differentially expressed in blood samples from RTT patients. Our results identify potential miRNA biomarkers selectively sorted into ADEVs and implicate RNA binding protein sequence dependent mechanisms for miRNA cargo loading.
Acetyl transferase EP300 deficiency leads to chronic replication stress mediated by defective fork protection at stalled replication forks
Authors: Barreto-Galvez Angelica , Niljikar Mrunmai , Gagliardi Julia , Zhang Ranran , Kumar Vasudha , Juruwala Aastha , Pradeep Archana , Shaikh Anam , Tiwari Priyanka , Sharma Kritika , Gerhardt Jeannine , Cao Jian , Kataoka Keisuke , Durbin Adam , Qi Jun , Ye B. Hilda , Madireddy Advaitha
Published: April 2023

Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a model to study EP300 deficiency and an p300-selective PROTAC degrader, degrader as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, accumulation of cytosolic DNA and transmission of unrepaired inherited DNA lesions in the subsequent G1-phase in EP300-deficient cells. We demonstrate that the DNA replication dynamics of EP300-mutated cells ATLL cells recapitulate features of BRCA-deficient cancers. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress and defective replication fork restart results in persistent genomic instability that underlie aggressive chemo-resistant tumorigenesis in humans.
Cardioprotective Activities ofArtemisia lactifloraExtract in Rat Cardiomyoblast (H9c2) Under Inflmmatory Sepsis
Authors: Kooltheat Nateelak , Chaiissarapap Amethyst , Jienthaisong Onanong , Mohlek Nutchanok , Kawinwanalai Wisa , Nokkaew Nuttikarn
Published: April 2023

Withdrawal Statement

The authors have withdrawn their manuscript owing to add more results. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs
Authors: Pornnoppadol Ghasidit , Bond Layne G. , Lucas Michael J. , Zupancic Jennifer M. , Kuo Yun-Huai , Zhang Boya , Greineder Colin F. , Tessier Peter M.
Published: April 2023

The inability of antibodies and other biologics to penetrate the blood-brain barrier (BBB) is a key limitation to their use in diagnostic, imaging, and therapeutic applications. One promising strategy is to deliver IgGs using a bispecific BBB shuttle, which involves fusing an IgG with a second affinity ligand that engages a cerebrovascular endothelial target and facilitates transport across the BBB. Nearly all prior efforts have focused on the transferrin receptor (TfR-1) as the prototypical endothelial target despite inherent delivery and safety challenges. Here we report bispecific antibody shuttles that engage CD98hc (also known as 4F2 and SLC3A2), the heavy chain of the large neutral amino acid transporter (LAT1), and efficiently transport IgGs into the brain parenchyma. Notably, CD98hc shuttles lead to much longer-lived brain retention of IgGs than TfR-1 shuttles while enabling more specific brain targeting due to limited CD98hc engagement in the brain parenchyma. We demonstrate the broad utility of the CD98hc shuttles by reformatting three existing IgGs as CD98hc bispecific shuttles and delivering them to the mouse brain parenchyma that either agonize a neuronal receptor (TrkB) or target other endogenous antigens on specific types of brain cells (neurons and astrocytes).
Structural basis for excitatory neuropeptide signaling
Authors: Kalienkova Valeria , Dandamudi Mowgli , Paulino Cristina , Lynagh Timothy
Published: April 2023

Rapid chemo-electric signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain1. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily, which occurs throughout the animal kingdom, is unique in its diversity of gating stimuli, with some DEG/ENaCs gated by conventional ligands such as neuropeptides, and others gated by e.g. pH, mechanical force, or enzymatic activity2-5. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. We have therefore dissected the structural basis for neuropeptide binding and gating in a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid wormMalacoceros fuliginosus6, using cryo-electron microscopy. High-resolution structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes that mediate channel gating. Complementary mutagenesis experiments confirm the functional roles of particular amino acid residues implicated by the structures. Our results illuminate ligand-induced channel gating in DEG/ENaCs and offer a structural template for the experimental dissection of channel pharmacology and ion conduction in a characteristically metazoan ion channel superfamily.
Complex synaptic and intrinsic interactions disrupt input/output functions in the hippocampus ofScn1bknockout mice
Authors: Chancey Jessica Hotard , Ahmed Alisha A. , Guillén Fernando Isaac , Howard MacKenzie A.
Published: April 2023

Mutations in theSCN1Bgene have been linked to severe developmental epileptic encephalopathies including Dravet syndrome.Scn1bknockout (KO) mice modelSCN1Bloss of function disorders, demonstrating seizures, developmental delays, and early death.SCN1Bencodes the protein β1, an ion channel auxiliary subunit that also has roles in cell adhesion, neurite outgrowth, and gene expression. The goal of this project is to better understand of how loss of β1 alters information processing in the brain, resulting in seizures and associated cognitive dysfunction. Using slice electrophysiology in the CA1 region of the hippocampus from male and femaleScn1bKO mice andwild-type (WT) littermates, we found that processing of physiologically relevant patternedSchaffercollateral (SC) stimulation produces larger, prolonged depolarizations and increased spiking in KO neurons compared to WTs. KO neurons exhibit enhanced intrinsic excitability, firing more action potentials with current injection. Interestingly, SC stimulation produces smaller, more facilitating excitatory and inhibitory postsynaptic currents in KO pyramidal neurons, but larger postsynaptic potentials with the same stimulation. We also found reduced intrinsic firing of parvalbumin-expressing interneurons and disrupted recruitment of both parvalbumin- and somatostatin-expressing interneurons in response to patterned synaptic stimulation. Neuronal information processing relies on the interplay between synaptic properties, intrinsic properties that amplify or suppress incoming synaptic signals, and firing properties that produce cellular output. We found changes at each of these levels inScn1bKO pyramidal neurons, resulting in fundamentally altered information processing in the hippocampus that likely contributes to the complex phenotypes ofSCN1B-linked epileptic encephalopathies.

Significance statement

Genetic developmental epileptic encephalopathies have limited treatment options, in part due to our lack of understanding of how genetic changes result in dysfunction at the cellular and circuit levels.SCN1Bis a gene linked to Dravet syndrome and other epileptic encephalopathies, andScn1bknockout mice phenocopy the human disease, allowing us to study underlying neurophysiological changes. Here we found changes at all levels of neuronal information processing in brains lacking β1, including intrinsic excitability, synaptic properties, and synaptic integration, resulting in greatly enhanced input/output functions of the hippocampus. Our study shows that loss of β1 results in a complex array of cellular and network changes that fundamentally alters information processing in the hippocampus.
Descending GABAergic Neurons of the RVM That Mediate Widespread Bilateral Antinociception
Authors: Ganley Robert P. , Magalhaes de Sousa Marília , Ranucci Matteo , Werder Kira , Öztürk Tugce , Wildner Hendrik , Zeilhofer Hanns Ulrich
Published: April 2023

Summary

Neurons projecting from the rostral ventromedial medulla (RVM) to the spinal dorsal horn are critical elements of endogenous pain control systems. Here, we describe a GABA/glycinergic pathway that predominantly innervates the superficial dorsal horn. Anatomical and optogenetic tracing of these neurons from a single unilateral site of the lumbar spinal cord indicated that these neurons give rise to a dense bilateral innervation of the spinal cord along its entire rostrocaudal axis. Chemogenetic activation of these neurons caused a bilateral and widespread reduction in heat, cold, and mechanical sensitivity, while their silencing with tetanus toxin induced allodynia and spontaneous pain-like aversive behaviors. Consistent with a continuous role in the prevention of spontaneous pain, many descending RVM GABAergic neurons were found to be tonically active. This pathway may therefore be relevant for widespread conditioned analgesia, while its dysfunction may underlie chronic widespread pain syndromes.