bluuebunny/crossref_metadata_2025 huggingface dataset:
Robust and cost-efficient single-cell sequencing through combinatorial pooling
Authors: Gawron Johannes , Cunha Luís , Borgsmüller Nico , Beerenwinkel Niko
Published: November 2024

Single-cell sequencing is widely used to study molecular cell-to-cell heterogeneity. Even though the cost of sequencing has dropped throughout the last decades, single-cell assays remain expensive, because they require strategies to index molecules by cells. The high costs of indexing can be mitigated by pooling samples prior to sequencing library preparation. Computational methods have been developed to leverage molecular features that are distinct between different samples to separate the pools into distinct datasets. However, since all multiplexed samples are processed in the same way, information on the origin of each demultiplexed dataset is lost. To map datasets to their sample of origin, additional information such as molecular indexing or additional genotyping is needed. Here, we propose a class of experimental designs that allows identifying the sample of origin of each demultiplexed dataset, only relying on the genetic profiles of the samples and the composition of pools. Our approach is based on splitting and pooling samples in specific combinations. We find a most cost-efficient experimental design in this class and prove its optimality. We present a dynamic programming algorithm to iteratively simplify an optimal experimental design by breaking it into several independent designs while maintaining optimality. Furthermore, we propose a subclass of experimental designs which allow robust sample identification even under partial failure of the experiment and present a provably optimal design in this subclass. We provide an implementation for automatic sample identification under these optimal combinatorial pooling strategies and demonstrate its functionality in a simulation study.
Estimating hierarchicalF–statistics from Pool–Seq data
Authors: Gautier Mathieu , Coronado-Zamora Marta , Vitalis Renaud
Published: November 2024

Introduced over seventy years ago,F–statistics have been and remain central to population and evolutionary genetics. Among them,FSTis one of the most commonly used descriptive statistics in empirical studies, notably to characterize the structure of genetic polymorphisms within and between populations, to shed light on the evolutionary history of populations, or to identify marker loci under differential selection for adaptive traits. However, the use ofFSTin simplified population models can overlook important hierarchical structures, such as geographic or temporal subdivisions, potentially leading to misleading interpretations and increasing false positives in genome scans for adaptive differentiation. HierarchicalF–statistics have been introduced to account for multiple predefined levels of population structure. Several estimators have also been proposed, including robust ones implemented in the popular R packagehierfstat. Nevertheless, these were primarily designed for individual genotyping data and can be computationally intensive for large genomic datasets. In this study, we extend previous work by developing unbiased method-of-moments estimators for hierarchicalF–statistics tailored for Pool–Seq data, a cost-effective alternative to individual genome sequencing. These Pool–Seq estimators have been developed in ananovaframework, using definitions based on identity-in-state probabilities. The new estimators have been implemented in an updated version of the R packagepoolfstat, together with estimators for sample allele count data derived from individual genotyping data. We validate and compare the performance of these estimators through extensive simulations under a hierarchical island model. Finally, we apply these estimators to real Pool–Seq data fromDrosophila melanogasterpopulations, demonstrating their usefulness in revealing population structure and identifying loci with high differentiation within or between groups of subpopulations and associated with spatial or temporal genetic variation.
Assessing changes in whole-brain structural connectivity in the unilateral 6-hydroxydopamine rat model of Parkinson’s Disease using diffusion imaging and tractography
Authors: Moshchin Mikhail , Schultz Roger J , Cheng Kevin P. , Osting Susan , Koeper James , Laluzerne Matthew , Trevathan James K. , Brzeczkowski Andrea , Luu Cuong P. , Yu John-Paul J , Betzel Richard , Lake Wendell B. , Hurley Samuel A. , Ludwig Kip A. , Suminski Aaron J.
Published: November 2024

I.

Parkinson’s disease (PD) is a multifactorial, progressive neurodegenerative disease that has a profound impact on those it afflicts. Its hallmark pathophysiology is characterized by degeneration of dopaminergic neurons in the midbrain which trigger a host of motor and non- motor symptoms. Many preclinical research efforts utilize unilateral lesion models to assess the neural mechanisms of PD and explore new therapeutic approaches because these models produce similar motor symptoms to those of PD patients. The goal of this work is to examine changes in brain structure resulting from a unilateral lesion both within the nigrostriatal system, where dopaminergic neurons are lost, and throughout the brain. Using multi-shell diffusion magnetic resonance imaging and correlational tractography, we assessed microstructural changes throughout the brain resulting from unilateral injection of 6-hydroxydopamine (6-OHDA) in the median forebrain bundle (MFB). Following lesioning, the PD phenotype was confirmed using behavioral and histological assessment. Correlational tractography found networks of fiber tracts that were either positively or negatively correlated with lesion status throughout the brain. Analyzing patterns of intra- and inter-hemispheric connectivity between the positively and negatively correlated fiber tracts revealed two separate neural networks. The first contained only negatively correlated fibers in the lesioned hemisphere consistent with the local effects of the lesion (i.e. dopaminergic depletion in the nigrostriatal system). The second contained systematically overlapping fiber tracts in the lesioned and non-lesioned hemispheres including the olfactory system and cerebellum, which we suggest are indicative of adaptive mechanisms to compensate for the lesion. Taken together, these results suggest that correlational tractography is a reasonable tool to examine whole brain structural changes in rodent models of neurodegenerative disease, and may have future translational value as a diagnostic tool for patients with PD.
New algorithms for unsupervised cell clustering from scRNA-seq data
Authors: Robles Melissa , Díaz-Riaño Jorge , Forigua Cristhian , Ojeda Sebastian , Guio Laura , Siaucho Paula , Guzman-Porras Jennifer , García-Orjuela Danilo , Naranjo Andres , Maradei Silvia , Quiroz Adolfo , Duitama Jorge
Published: November 2024

The identification of cell types is a basic step of the pipeline for Single-Cell RNA sequencing data analysis. However, unsupervised clustering of cells from scRNA-seq data has multiple challenges: the high dimensional nature of the data, the sparse nature of the gene expression matrix, and the presence of technical noise that can introduce false zero entries. In this study, we introduce new algorithms for clustering scRNA-seq data. The first algorithm builds ak-MST graph from distances obtained directly from the input data without dimensionality reduction. The computation follows an iterative procedure of k steps in which each step calculates and stores the edges of minimum spanning trees over different subgraphs obtained removing edges selected in previous iterations. The Louvain algorithm is executed on thek-MST graph for cell clustering. We also explored alternatives based on neural networks in which an autoencoder is used to learn the parameters of a Gaussian mixture model, aiming to improve the handling of clusters with different shapes and sizes. Benchmark experiments with simulated data and public datasets show that the algorithms proposed in this work have competitive accuracy, compared to previous solutions, but also that sequencing depth, number of cells and tissue types have important effects on the performance of the algorithms. Moreover, we performed further experiments with scRNA-data taken from a patient with refractory epilepsy. The AE-GMM model achieved the best accuracy for this dataset, and thek-MST ranked first among methods that do not require previous information on the expected number of clusters.
STEP: Deciphering Spatial Atlas at Single-Cell Level with Whole-Transcriptome Coverage
Authors: Hu Zheqi , Zhu Zirui , Cai Linghan , Zhan Yangen , Yan Xiangming , Chen Jingyun , Sun Bingqian , Du Sijing , Jiang Songhan , Wang Hongpeng , Zhang Yongbing
Published: November 2024

Recent advances in spatial transcriptomics have revolutionized our understanding of tissue spatial architecture and biological processes. However, many of these technologies face significant challenges in achieving either single-cell resolution or comprehensive whole-transcriptome profiling, hindering their capacity to fully elucidate intercellular interactions and the tissue microenvironment. To address these limitations, we present STEP, a hybrid framework that synergistically integrates probabilistic models with deep learning techniques for spatial transcriptome analysis. Through innovations in model and algorithm design, STEP not only enhances sequencing-based spatial transcriptome data to single-cell resolution but accurately infers transcriptome-wide expression levels for image-based spatial transcriptomic. By leveraging the nuclear features extracted from histological images, STEP achieves precise predictions of cell type and gene expression and effectively diffuses the discriminative ability to serial sections for modeling solid tissue landscapes. The capability is particularly advantageous for analyzing cells with distinctive characteristics, such as cancer cells, enabling cross-sample inference. In addition, STEP simulates intercellular communication through a spatially resolved cell-cell interaction network, uncovering intrinsic biological processes. Overall, STEP equips researchers with a powerful tool for understanding biological functions and unveiling spatial gene expression patterns, paving the way for advancements in spatial transcriptomics research. Code is available athttps://github.com/childishHU/STEP.
Higher-Order Interaction Analysis via Hypergraph Models for Studying Multidimensional Neuroscience Data
Authors: Bilbao Dalma , Aimar Hugo , Torterolo Pablo , Mateos Diego M.
Published: November 2024

Higher-Order Interaction (HOI) theory offers a powerful framework for capturing complex, non-linear relationships within multidimensional systems, moving beyond traditional pairwise graph methods to encompass multi-way interactions. This study applies HOI analysis, specifically using hypergraph theory, to explore intricate connectivity patterns in electrophysiological signals from neuroscience. Hypergraphs were constructed from connectivity data across various frequency bands, characterized through metrics such as spectral entropy, hyperedge centrality, and vertex centrality, and compared using spectral and centrality distance measures. Three distinct neurophysiological datasets were analyzed: intracranial EEG signals from rats during different sleep stages, scalp EEG data to distinguish between epilepsy types, and MEG recordings of seizure dynamics. The findings highlight the effectiveness of hypergraph-based HOI analysis in mapping neural dynamics across normal and pathological brain states. In sleep studies, it reveals distinct connectivity patterns between REM and NREM stages, while in epilepsy, it differentiates seizure types and stages, identifying spectral entropy as a potential marker for seizure onset. Notably, HOI analysis captures differences between primary and secondary generalized epilepsy, suggesting enhanced diagnostic accuracy. This approach provides a powerful tool for understanding complex neural interactions in high-dimensional data.
Large-scale Signal Propagation Modes in the Human Brain
Authors: Song Youngjo , Kim Pyeong Soo , Philip Benjamin A. , Kim Taewon
Published: November 2024

The brain’s large-scale temporal dynamics play a crucial role in understanding its operations, but developing a cohesive framework to integrate the potentially extensive array of spatiotemporal patterns remains elusive. Our work addresses this gap by identifying multiple large-scale signal propagation modes in resting-state fMRI time series under a unified methodological framework. We found five distinct modes that effectively predict future blood-oxygen-level-dependent (BOLD) signal dynamics, each reconciling transitions between well-known large-scale brain networks into coherent spatiotemporal units. By utilizing these coherent units, our approach circumvents the need to explore combinatorial explosion of transitions between potential states, enabling parsimonious modeling and effective prediction of whole-brain temporal evolution. Each mode captures specific operational dimensions of neural resource allocation, ensuring their interpretability. Importantly, we showed that complex spatiotemporal features emerge from the superposition of these few propagation modes, unifying a broad spectrum of well-known brain dynamics phenomena. Our results lay the groundwork for a unified framework to understand large-scale spatiotemporal brain organization. Moreover, individual differences in mode expression profiles correlate with general cognitive abilities, exhibit heritability, and demonstrate cross-task stability, underscoring their functional significance. This could lead to efficient methods for characterizing functional fingerprints and advancing diagnostic approaches for neurological disorders.
The Rise of Polyploids During Environmental Catastrophes
Authors: Chen Hengchi , Almeida-Silva Fabricio , Logghe Garben , Bonte Dries , Van de Peer Yves
Published: November 2024

Polyploidy, or whole-genome duplication (WGD), both a strong evolutionary and ecological force and a potential pitfall, occurs extensively across the tree of life, particularly in angiosperms. While polyploid organisms are prevalent, ancient polyploidy (paleopolyploidy) is decisively rare. Numerous studies have revealed adaptive changes at the genomic, cellular, and physiological levels that confer evolutionary advantages to polyploids, but the factors contributing to their long-term success remain poorly understood. Here, we assembled a large angiosperm genome dataset encompassing 470 different angiosperm species. We reconstructed a highly concordant evolutionary timescale of angiosperms and conducted a large-scale study to identify and date WGDs across this set of species. We unveiled 132 independent ancient WGD events that do not seem randomly distributed in time, but clustered around periods of great environmental upheaval. We observed that a majority of paleopolyploidizations occurred between 50 and 75 mya, coinciding with the Paleocene-Eocene Thermal Maximum and the Cretaceous-Paleogene (K-Pg) boundary. A more recent wave of ancient WGDs can be observed between 25 and 40 mya, coinciding with periods of glaciation expansion, rapid shifts in sea surface temperatures, and a marked decrease in global mean surface temperature. Integrating different lines of evidence, we argue that polyploids have an increased chance of survival during times of great environmental turmoil.
Mitochondrial ROS and HIF-1α signalling mediate critical period plasticity
Authors: Sobrido-Cameán Daniel , Coulson Bramwell , Miller Michael , Oswald Matthew C.W. , Pettini Tom , Baines Richard A. , Landgraf Matthias
Published: November 2024

As developing networks transition from spontaneous irregular to patterned activity, they undergo plastic tuning phases, termed “critical periods”; “critical” because disturbances during these phases can lead to lasting changes in network development and output. Critical periods are common to developing nervous systems, with analogous features shared from insects to mammals, yet the core signalling mechanisms that underly cellular critical period plasticity have remained elusive. To identify these, we exploited theDrosophilalarval locomotor network as an advantageous model system. It has a defined critical period and offers unparalleled access to identified network elements, including the neuromuscular junction as a model synapse. We find that manipulations of a single motoneuron or muscle cell during the critical period lead to predictable, and permanent, cell-specific changes. This demonstrates that critical period adjustments occur at a single cell level. Mechanistically, we identified mitochondrial reactive oxygen species (ROS) as causative. Specifically, we show that ROS produced by complex I of the mitochondrial electron transport chain, generated by the reverse flow of electrons, are necessary and instructive for critical period-regulated plasticity. Downstream of ROS, we identified theDrosophilahomologue of hypoxia inducible factor (HIF-1α), as required for transducing the mitochondrial ROS signal to the nucleus. This signalling axis is also sufficient to cell autonomously specify changes in neuronal properties and animal behaviour but, again, only when activated during the embryonic critical period. Thus, we have identified specific mitochondrial ROS and HIF-1α as primary signals that mediate critical period plasticity.
Effects of sex, mating status, and genetic background on circadian behavior inDrosophila
Authors: Akpoghiran Oghenerukevwe , Strich Alexandra K. , Koh Kyunghee
Published: November 2024

Circadian rhythms play a crucial role in regulating behavior, physiology, and health. Sexual dimorphism, a widespread phenomenon across species, influences circadian behaviors. Additionally, post-mating physiological changes in females are known to modulate various behaviors, yet their effects on circadian rhythms remain underexplored. Here, usingDrosophila melanogaster, a powerful model for studying circadian mechanisms, we systematically assessed the impact of sex and mating status on circadian behavior. We measured circadian period length and rhythm strength in virgin and mated males and females, including females mated to males lacking Sex Peptide (SP), a key mediator of post-mating changes. Across four wild-type and control strains, we found that males consistently exhibited shorter circadian periods than females, regardless of mating status, suggesting that circadian period length is a robust sexually dimorphic trait. In contrast, rhythm strength was influenced by the interaction between sex and mating status, with female mating generally reducing rhythm strength in the presence of SP signaling. Notably, genetic background significantly modulated these effects on rhythm strength. Our findings demonstrate that while circadian period length is a stable sex-specific trait, rhythm strength is shaped by a complex interplay between sex, mating status, and genetic background. This study advances our understanding of how sex and mating influence circadian rhythms inDrosophilaand provides a foundation for future research into sexually dimorphic mechanisms underlying human diseases associated with circadian disruptions.
Sex-specific perturbations of neuronal development caused by mutations in the autism risk geneDDX3X
Authors: Mossa Adele , Dierdorff Lauren , Lukin Jeronimo , Park Yeaji , Fiorenzani Chiara , Akpinar Zeynep , Garcia-Forn Marta , De Rubeis Silvia
Published: November 2024

DDX3Xis an X-linked RNA helicases that escapes X chromosome inactivation and is expressed at higher levels in female brains. Mutations inDDX3Xare associated with intellectual disability (ID) and autism spectrum disorder (ASD) and are predominantly identified in females. Using cellular and mouse models, we show thatDdx3xmediates sexual dimorphisms in brain development at a molecular, cellular, and behavioral level. During cortical neuronal development,Ddx3xsustains a female-biased signature of enhanced ribosomal biogenesis and mRNA translation. Female neurons display higher levels of ribosomal proteins and larger nucleoli, and these sex dimorphisms are obliterated byDdx3xloss.Ddx3xregulates dendritic outgrowth in a sex- and dose-dependent manner in both female and male neurons, and dendritic spine development only in female neurons. Further, ablatingDdx3xconditionally in forebrain neurons is sufficient to yield sex-specific changes in developmental outcomes and motor function. Together, these findings poseDdx3xas a mediator of sexual differentiation during neurodevelopment and open new avenues to understand sex differences in health and disease.
Alanine-scanning of the yeast killer toxin K2 reveals key residues for activity, gain-of-function variants, and supports prediction of precursor processing and 3D structure
Authors: Prins Rianne C. , Marinus Tycho , Dafni Eyal , Yacoby Iftach , Billerbeck Sonja
Published: November 2024

Yeast killer toxins (YKTs) are antimicrobial proteins secreted by yeast with potential applications ranging from food preservation to therapeutic agents in human health. However, the practical use of many YTKs is limited by specific pH requirements, low temperature stability, low production yields, and narrow target specificity. While protein engineering could potentially overcome these challenges, progress is hindered by a lack of detailed knowledge about sequence-function relationships and structural data for these often multi-step processed proteins. In this study, we focused on the YKT K2, encoded by the M2 dsRNA satellite virus inSaccharomyces cerevisiae. Using alanine scanning mutagenesis of the full open reading frame and structure predictions combined with molecular dynamics simulations, we generated a comprehensive sequence-function map, refined the model for the proteolytic processing of the K2 precursor, and predicted the mature toxin structure. Our findings also demonstrate that K2 can be engineered towards enhanced toxicity and altered target specificity through single-site mutations. Furthermore, we identified structural homology between K2 and the SMK toxin from the yeastMillerozyma farinosa. Our cost-effective workflow provides a platform to broadly map YKT sequence-structure-function relationships, facilitating the engineering towards toxin-based technologies. The workflow could also serve as a template to resolve the processing and conformations of other proteins within the secretory pathway – a dynamic multi-step process that is challenging to structurally capture by purification and solving structures of intermediates.
14-3-3 binding maintains the Parkinson’s associated kinase LRRK2 in an inactive state
Authors: Martinez Fiesco Juliana A. , Li Ning , Alvarez de la Cruz Astrid , Metcalfe Riley D. , Beilina Alexandra , Cookson Mark R. , Zhang Ping
Published: November 2024

Leucine-rich repeat kinase 2 (LRRK2) is a central player in cellular signaling and a significant contributor to Parkinson’s disease (PD) pathogenesis. 14-3-3 proteins are essential regulators of LRRK2, modulating its activity. Here, we present the cryo- electron microscopy structure of the LRRK2:14-3-32autoinhibitory complex, showing that a 14-3-3 dimer stabilizes an autoinhibited LRRK2 monomer by binding to key phosphorylation sites and the COR-A and COR-B subdomains within the Roc-COR GTPase domain of LRRK2. This interaction locks LRRK2 in an inactive conformation, restricting LRR domain mobility and preventing dimerization and oligomer formation. Our mutagenesis studies reveal that PD-associated mutations at the COR:14-3-3 interface and within the GTPase domain reduce 14-3-3 binding, diminishing its inhibitory effect on LRRK2. These findings provide a structural basis for understanding how LRRK2 likely remains dormant within cells, illuminate aspects of critical PD biomarkers, and suggest therapeutic strategies to enhance LRRK2-14-3-3 interactions to treat PD and related disorders.
Single CAR-Dual target: Intracranial Delivery of Anti-PD-L1 CAR T Cells Effectively Eradicates Glioma and Immunosuppressive Cells in the Tumor Microenvironment
Authors: Ulloa-Navas Maria J. , Luo Yan , Sanchez-Garavito Jesus E , Qie Yaqing , Jones Vanessa K , Brooks Mieu M , Aramburu-Berckemeyer Manuela , Hundal Tanya , Gadd Martha E. , Gomez-Palmero Cristina , Brim Andrew D. , Rivera-Valentin Rocio , Basil Aleeshba N. , Tutor-New Frederick Q. , Hirdler Jacob B. , Irani Sarosh R , Dronca Roxana , Kharfan-Dabaja Mohamed A , Ren Yingxue , Dong Haidong , Qin Hong , Quinones-Hinojosa Alfredo
Published: November 2024

With the goal to overcome the limited treatment options and poor prognosis of glioblastoma (GBM), we have developed a PD-L1-targeting CAR T cell therapy, MC9999. In vitro experiments with MC9999 CAR T cells derived from GBM patients exhibited potent, antigen-specific cytotoxicity against autologous tumor cells and immunosuppressive cells within the tumor microenvironment (TME). In an orthotopic GBM model using patient-derived brain tumor-initiating cells, the intracranial delivery of MC9999 CAR T cells eradicated established tumors and improved survival. Single-cell RNA sequencing indicated that MC9999 CAR T cells activate interferon pathways, leading to GBM cell apoptosis. Multi-immunohistochemistry confirmed localized PD-L1 expression on tumor cells and TME-residing macrophages, but not in neurons or glia in patient tissue. The local delivery of MC9999 CAR T cells may be a safe, effective approach for simultaneously targeting PD-L1-positive GBM and its immunosuppressive TME and a strategy to overcome immune evasion and enhance the therapeutic potency of CAR T cell therapy against GBM.
Neuronal heterogeneity of normalization strength in a circuit model
Authors: Song Deying , Ruff Douglas , Cohen Marlene , Huang Chengcheng
Published: November 2024

The size of a neuron’s receptive field increases along the visual hierarchy. Neurons in higher-order visual areas integrate information through a canonical computation called normalization, where neurons respond sublinearly to multiple stimuli in the receptive field. Neurons in the visual cortex exhibit highly heterogeneous degrees of normalization. Recent population recordings from visual cortex find that the interactions between neurons, measured by spike count correlations, depend on their normalization strengths. However, the circuit mechanism underlying the heterogeneity of normalization is unclear. In this work, we study normalization in a spiking neuron network model of visual cortex. The model produces a range of neuronal heterogeneity of normalization strength and the heterogeneity is highly correlated with the inhibitory current each neuron receives. Our model reproduces the dependence of spike count correlations on normalization as observed in experimental data, which is explained by the covariance with the inhibitory current. We find that neurons with stronger normalization are more sensitive to contrast differences in images and encode information more efficiently. In addition, networks with more heterogeneity in normalization encode more information about visual stimuli. Together, our model provides a mechanistic explanation of heterogeneous normalization strengths in the visual cortex, and sheds new light on the computational benefits of neuronal heterogeneity.
The pentameric chloride channel BEST1 is activated by extracellular GABA
Authors: Pant Swati , Tam Stephanie W. , Long Stephen B.
Published: November 2024

Bestrophin 1 (BEST1) is chloride channel expressed in the eye, central nervous system (CNS), and other tissues in the body. A link between BEST1 and the principal inhibitory neurotransmitter γ-aminobutyric acid (GABA) has been proposed. The most appreciated receptors for extracellular GABA are the GABABG-protein coupled receptors and the pentameric GABAAchloride channels, both of which have fundamental roles in the CNS. Here, we demonstrate that BEST1 is directly activated by GABA. Through functional studies and atomic-resolution structures of human and chicken BEST1, we identify a GABA binding site on the channel’s extracellular side and determine the mechanism by which GABA binding induces opening of the channel’s central gate. This same gate is activated by intracellular [Ca2+], indicating that BEST1 is controlled by ligands from both sides of the membrane. The studies demonstrate that BEST1, which shares no structural homology with GABAA, is a GABA-activated chloride channel. The physiological implications of this finding remain to be studied.

Significance Statement

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system. Extracellular GABA is primarily sensed by GABABG-protein coupled receptors, and GABAApentameric chloride channels. We show that the chloride channel bestrophin-1 (BEST1) is also activated by extracellular GABA. The application of GABA, but not glycine or other endogenous molecules we tested, markedly potentiates chloride currents through the channel. Structural studies combined with electrophysiology and other functional studies reveal the mechanism of GABA activation. GABA binding within the outer entryway of the channel allosterically controls its gate, ‘the neck’. Molecular interactions with GABA resemble those in GABAAreceptors, despite a lack of homology between the channels. The physiological significance of GABA-activation in BEST1 remains to be studied.
A modular gate system for autonomous control of rodent behavior
Authors: Lester Adam W. , Kaur Gurnoor , Djafri Nadira , Madhav Manu S.
Published: November 2024

Rodent mazes have been used for decades to study the neural basis of behavior. Advancements in rapid prototyping techniques and access to affordable electronics allows laboratories with sufficient expertise in engineering and programming to customize and construct maze apparatuses and behavioral tasks, thereby increasing the ability of their studies to answer specific scientific questions. We designed and built a rodent gate system that lowers this bar of expertise even further. The NC4gate system is a robust mechanical design that can be built using low-cost hardware and execute thousands of cycles before maintenance. Up to 512 gates can be controlled using a single computer. Users can control the gates interactively using a Python-based graphical interface and programmatically using an extensible API. We hope that the open-source hardware / software and extensive documentation enables laboratories to build these affordable and robust gates and seamlessly incorporate automatic behavior control into their existing or new rodent tasks.

Significance Statement

Rodent mazes are used by thousands of laboratories and research institutions across the world to study learning and memory, as well as the effects of pharmacological, genetic and environmental manipulations. Ideally, maze and task designs should be customized to the scientific questions at hand. It is challenging, however, for many laboratories to build, program, and operate custom mazes, requiring them instead to rely on expensive and proprietary commercial solutions. The most complex components of most mazes are moving gates that restrict and direct rodent behavior. Here we provide the open-source hardware and software for a gate system that is extensible, affordable and robust, removing this critical barrier to customized mazes.
A Multi-Strain, Biofilm-Forming Cocktail ofBacillusspp. andPediococcusspp. Alters the Microbial Composition on Polyethylene Calf Housing Surfaces
Authors: Reynolds C.A. , Scuderi R.A. , Skidmore A.L. , Duniere L. , Morrison S.Y.
Published: November 2024

Application of a beneficial microbial cocktail ofBacillusspp. andPediococcusspp. was evaluated first for adherence to polyethylene calf hutch material, and second, to determine if application in situ to individual calf hutches post-cleaning influenced surface recolonization by enteric pathogens. Three treatments were utilized: 1) no application (NC), 2) chlorine-free, distilled water (DW), or 3) an application of a microbial inoculant containingBacillusspp. andPediococcusspp. at a concentration of 0.4 g/m2of hutch space (LF). Thirty-six 15 × 15 cm pieces of naïve, sterile polyethylene calf hutch material received either NC or LF, were incubated at 28°C, and bacterial growth was evaluated by total aerobic plate counts at 24, 48, and 72 h post-application. Thirty polyethylene calf hutches (n = 10/treatment) were randomized to either NC, DW, or LF 24 h after cleaning. Calves were placed in the hutches 24 h after treatment application and monitored daily for 28 d. In situ surface samples were randomized by time from five unique locations within the calf hutch interior: 24 h post-cleaning, then 24 h, 7 d, 14 d, and 21 d post-application. Total aerobic plate counts and culture-independent approaches RT-qPCR and 16S amplicon sequencing were used to detect and identify the composition of the bacterial community in situ. The bacteria in the inoculant were able to successfully colonize on polyethylene, and application to individual polyethylene calf housing in situ influenced microbial diversity and reduced the presence of some undesirable bacteria on high-contact interior surfaces.

IMPORTANCE

Due to its multifactorial nature, neonatal calf diarrhea can be difficult to manage on farms. Clean housing environments are a critical disease control point, especially for calves less than one month of age. Application of a beneficial biofilm-forming bacterial product after cleaning of neonatal calf housing may influence the microbial communities present on the surface, particularly those that may present disease risk to calves in early life.
Divergent Visuomotor Strategies in Teleosts: Neural Circuit Mechanisms in Zebrafish andDanionella cerebrum
Authors: Fouke Kaitlyn E. , He Zichen , Loring Matthew D. , Naumann Eva A.
Published: November 2024

Many animals respond to sensory cues with species-specific coordinated movements to successfully navigate their environment. However, the neural mechanisms that support diverse sensorimotor transformations across species with distinct navigational strategies remain largely unexplored. By comparing related teleost species, zebrafish (Danio rerio, ZF) andDanionella cerebrum(DC), we investigated behavioral patterns and neural architectures during the visually guided optomotor response (OMR). Closed-loop behavioral tracking during visual stimulation revealed that larvalZFemploy burst-and-glide locomotion, while larvalDCdisplay continuous, smooth swimming punctuated with sharp directional turns. AlthoughDCachieve higher average speeds, they lack the direction-dependent velocity modulation observed inZF. Whole-brain two-photon calcium imaging and tail tracking in head-fixed fish reveals that both species exhibit direction-selective motion encoding in homologous regions, including the retinorecipient pretectum, withDCexhibiting fewer binocular, direction-selective neurons overall. Kinematic analysis of head-fixed behavior reveals thatDCsustain significantly longer directed swim events across all stimuli thanZF, highlighting the divergent visuomotor strategies, withZFreducing tail movement duration in response to oblique, turn-inducing stimuli. Lateralized motor-associated neural activity in the medial and anterior hindbrain of both species suggests a shared circuit motif, with distinct neural circuits that independently control movement vigor and direction. These findings highlight the diversity in visuomotor strategies among teleost species, underscored by shared sensorimotor neural circuit motifs, and establish a robust framework for unraveling the neural mechanisms driving continuous and discrete visually guided locomotion, paving the way for deeper insights into vertebrate sensorimotor functions.

Research Highlights

Larval DCexhibit faster swimming thanZF, matching the direction of visual motion.

DCexecute OMR in smooth, curved swimming patterns, interspersed with sharp directional turns.

ZFandDCshare similar visuomotor neural architecture, recruiting pretectal and hindbrain regions.

ZFandDCdemonstrate lateralized encoding of turns, particularly in medial hindbrain neurons.

In Brief

LarvalDanionella cerebrumrespond to global visual motion cues in smooth, low-angle swimming patterns, interspersed with sharp directional turns, swimming consistently faster than zebrafish. Fouke et al. use behavioral tracking of freely moving and head fixed fish to reveal an evolutionarily conserved visuomotor neural architecture transforming visual motion cues into species-specific locomotor behaviors.Graphical Abstract
Advancing protein display on bacterial spores through an extensive survey of coat components
Authors: Chappell Todd C. , Nicks Trevor B. , Cheng Jan-Fang , Tarver Angela , Lee Jessica A. , Kalia Arushi , Nair Nikhil U.
Published: November 2024

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though theBacillus subtilisspore is composed of >40 proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SP investigated, 26 formed functional fusions with β-glucuronidase (GUS) – a ∼272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed the 1) SP promoter activation dynamics during growth, 2) supported GUS activity on spores, 3) surface availability, 4) protection from thermal and proteolytic degradation, and 5) compatibility of co-expression of many of these fusions. Multi-copy expression and pairwise co-expression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and difficulty in predicting compatibility between different SPs fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA (smallsporecoat assembly proteinA) as an effective spore display carrier. SscA supported enzyme activity at least fourfold higher than any other SP, including the well-established and popular anchor, CotY. We attribute this to the biochemical and genetic features of SscA; its promoter demonstrated early and sustained activation relative to other SPs and its small size (∼3 KDa) likely minimally interferes with enzyme folding, oligomerization, and activity. Although the specific localization of SscA within the spore coat has not been established, its hydrophobic nature and low surface availability suggests that it assembles internally within the spore coat, which makes it highly stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledgebase to advance the biotechnological utility ofB. subtilisspores.
Unbiased data-driven analysis of five amyloid-beta peptides for biomarker investigations in familial Alzheimer’s disease
Authors: Llorente-Saguer Isaac , Gabriele Rebecca , Bradshaw Teisha , Leckey Claire A. , Belder Christopher R.S. , Chávez-Gutiérrez Lucía , de Silva Rohan , Fox Nick C. , Wray Selina , Oxtoby Neil P. , Arber Charles
Published: November 2024

Structured Abstract

INTRODUCTION

Changes to the relative abundance of amyloid-beta (Aβ) peptides are hallmarks of Alzheimer’s disease (AD). iPSC-derived neurons offer a physiological model of Aβ production. We employed unbiased, data-driven analyses to investigate combinations of Aβ peptides as AD biomarkers and the relative contribution of peptides to AD pathogenesis.

METHODS

We measured Aβ37, Aβ38, Aβ40, Aβ42 and Aβ43 in ten iPSC-neuronal cultures fromPSEN1mutation carriers. We combined these data with published cell model data and used linear weighted combinations to 1) distinguish AD from controls, and 2) predict age-at-onset forPSEN1mutations.

RESULTS

Data-driven approaches distinguished Aβ42 and Aβ43 from shorter peptides, providing unbiased evidence for their contribution to disease. Weighted linear combinations of Aβ peptides outperform Aβ42/40 and provide insights into relative peptide contribution as biomarkers; the optimal ratio for all data is represented as (21 · Aβ37 + 10 · Aβ38 + 69 · Aβ40)/(94 · Aβ42 + 6 · Aβ43).

DISCUSSION

The algorithm discovered herein can be further refined to improve biomarkers for AD.
Visual speech enhances auditory onset timing and envelope tracking through distinct mechanisms
Authors: Cao Cody Zhewei , Stacey William C , Wasade Vibhangini S , Towle Vernon L , Tao James X , Wu Shasha , Issa Naoum P , Brang David
Published: November 2024

Seeing a speaker’s face facilitates speech recognition in challenging listening environments. Prior work has shown that visual speech contains timing information to aid auditory speech processing, yet how these signals are integrated within the auditory system during audiovisual speech perception remains poorly understood. Observation of preparatory mouth movements may initiate phase reset of intrinsic oscillations, potentially sensitizing the auditory system for receptive speech processing, while observation of mouth movements post speech onset may facilitate entrainment to the speech envelope. Yet, little work has been done to test whether visual speech enhances encoding of auditory speech onset, speech envelope tracking, or both, and through independent or overlapping mechanisms. To investigate this, we examined the ways in which visual speech timing information alters theta band power and phase using human intracranial electroencephalography (iEEG) recordings in a large group of patients with epilepsy (n= 21). Prior to speech onset, preparatory mouth movements elicited theta phase reset (increased inter-trial phase coherence; ITPC) throughout the superior temporal gyrus (STG), which is thought to enhance speech onset encoding. Following speech onset, visual speech modulated theta ITPC only at anterior STG electrodes while theta power was modulated at posterior STG electrodes. Pre- and post-speech onset were spatially and temporally dissociated, consistent with the hypothesis that audiovisual speech onset encoding and envelope tracking mechanisms are partially distinct. Crucially, congruent and incongruent visual speech, designed here to have identical visual timing information about speech onset time, but different visual mouth evolution, produced only a small difference in the phase of theta band oscillations in the anterior STG, highlighting a more restricted role of visual speech in ongoing auditory entrainment. These results support the hypothesis that visual speech improves the precision of auditory speech encoding through two separate mechanisms, with auditory speech onset encoded throughout the entire STG and ongoing speech envelope tracking within anterior STG.
Dengue Protease-Mediated Activation and Polarization of Macrophages
Authors: Misra Rajdip , Mukherjee Kaustuv , Karmakar Joyshree , Pal Uttam , Maiti Nakul C
Published: November 2024

Controlled activation of macrophages to achieve desired phenotypes in vivo and in vitro could lead to possible treatments for several inflammatory and proliferative diseases. This investigation uniquely established that dengue protease enhances the polarization and activation of human and murine macrophages following extracellular exposure. It was observed that macrophages have a robust response to extracellularly administered purified dengue NS2B/NS3 protease; it stimulated the macrophages in classical M1 directions and enhanced both the mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinases (JNK). The cells exhibited an increase in the intracellular levels of proinflammatory cytokines such as IL-2, IL-6, IL-12, and IFN-γ and an elevation of phosphorylated versions of Akt, P38 MARK, SAPK, and ERK. In tandem, this investigation also established that the heightened responses of stimulated cells were associated with an increase in the cellular reactive oxygen species (ROS) level and the nuclear translocation of the NF-κB (P65) protein. The cell viability assay showed that NS2B/NS3 protease exerts no major toxic effect on macrophages after 24 hours of treatment, even at a dosage (20 μg/ml) which was four times higher than the effective dose (5 μg/ml). Remarkably, we also observed that the native form of the viral protease, which drives its enzyme activities, had no bearing on the antigenic qualities of the enzyme. Thus, our study highlighted the efficacy of dengue viral NS2B/NS3 protease as a non-toxic ex vivo macrophage activating/polarizing agent and may serve a vital role in macrophage-based cell therapy in the near future.
Pharmacological Inhibition of SUMOylation with TAK-981 Mimics Genetic HypoSUMOylation in Murine Perigonadal White Adipose Tissue
Authors: Dufour Damien , Zhao Xu , Chaleil Florian , Nothnagel Patrizia MC , Bjoras Magnar , Lefrancois-Martinez Anne-Marie , Martinez Antoine , Chymkowitch Pierre
Published: November 2024

Post-translational modification by the small ubiquitin-like modifier (SUMO) is essential for cellular differentiation and homeostasis. Here, we investigate the role of SUMOylation in adipose tissue development using TAK-981, a pharmacological inhibitor of SUMOylation. Administration of TAK-981 to mice resulted in significant defect in weight gain and adipocyte atrophy in perigonadal white adipose tissue (gWAT) depots. Gene expression analyses revealed a marked downregulation of adipogenic genes, including Pparg, Cebpa, and Fasn. Our data thus indicate that TAK-981 treatment impaired adipogenesis in gWAT, consistent with prior findings that SUMOylation supports transcriptional regulation of adipogenesis and lipid metabolism. We also found significant infiltration of immune cells and efferocytosis in gWAT. Our results thus indicate that SUMOylation inhibition using a small molecule phenocopies genetic hypoSUMOylation models, highlighting its critical role in maintaining adipocyte functionality and immune environment. These findings provide evidence that SUMOylation is essential for fat accumulation in vivo. Furthermore, given that TAK-981 is currently under clinical evaluation for the treatment of solid tumors, our results underscore the importance of considering the potential unintended effects of SUMOylation inhibition on adipose tissue in patients.
Multi-level transcriptional regulation of embryonic sex determination and dosage compensation by the X-signal elementsex-1
Authors: Jash Eshna , Tan Zoey M. , Rakozy Audry I. , Azhar Anati Alyaa , Mendoza Hector , Csankovszki Györgyi
Published: November 2024

TheC. elegansnuclear hormone receptorsex-1is known to be an embryonic X-signal element that repressesxol-1, the sex-switch gene that is the master regulator of sex determination and dosage compensation. Several prior studies onsex-1function have suggested thatsex-1may have additional downstream roles beyond the regulation ofxol-1expression. In this study we characterize some of these additional roles ofsex-1in regulating the dual processes of sex determination and dosage compensation during embryogenesis. Our study reveals thatsex-1acts on many of the downstream targets ofxol-1in axol-1-independent manner. Further analysis of these shared but independently regulated downstream targets uncovered thatsex-1mediates the expression of hermaphrodite- and male-biased genes during embryogenesis. We validatedsex-1binding on one of these downstream targets, the male-developmental geneher-1.Our data suggests a model wheresex-1exhibits multi-level direct transcriptional regulation on several targets, includingxol-1and genes downstream ofxol-1,to reinforce the appropriate expression of sex-biased transcripts in XX embryos. Furthermore, we found thatxol-1 sex-1double mutants show defects in dosage compensation. Our study provides evidence that misregulation ofdpy-21, one of the components of the dosage compensation complex, and the subsequent misregulation of H4K20me1 enrichment on the X chromosomes, may contribute to this defect.