bluuebunny/crossref_metadata_2025_split huggingface dataset:
Accelerated protein retention expansion microscopy using microwave radiation
Authors: Bullard Meghan R. , Cervantes Juan Carlos Martinez , Quaicoe Norisha B. , Jin Amanda , Adams Danya A. , Lin Jessica M. , Iliadis Elena , Seidler Tess M. , Cervantes-Sandoval Isaac , He Hai-yan 
Published: May 2024

AbstractProtein retention expansion microscopy (ExM) retains genetically encoded fluorescent proteins or antibody-conjugated fluorescent probes in fixed tissue and isotropically expands the tissue through a swellable polymer network to allow nanoscale (<70 nm) resolution on diffraction-limited confocal microscopes. Despite numerous advantages ExM brings to biological studies, the full protocol is time-consuming and can take multiple days to complete. Here, we adapted the ExM protocol to the vibratome-sectioned brain tissue ofXenopus laevistadpoles and implemented a microwave-assisted protocol to reduce the workflow from days to hours. In addition to the significantly accelerated processing time, our microwave-assisted ExM (M/WExM) protocol maintains the superior resolution and signal-to-noise ratio of the original ExM protocol. Furthermore, theM/WExM protocol yields higher magnitude of expansion, suggesting that in addition to accelerating the process through increased diffusion rate of reagents, microwave radiation may also facilitate the expansion process. To demonstrate the applicability of this method to other specimens and protocols, we adapted the microwave-accelerated protocol to whole mount adult brain tissue ofDrosophila melanogasterfruit flies, and successfully reduced the total processing time of a widely-usedDrosophilaIHC-ExM protocol from 6 days to 2 days. Our results demonstrate that with appropriate adjustment of the microwave parameters (wattage, pulse duration, interval, and number of cycles), this protocol can be readily adapted to different model organisms and tissue types to greatly increase the efficiency of ExM experiments.
Antibiotic treatment induces microbiome dysbiosis and reduction of neuroinflammation following traumatic brain injury in mice
Authors: Flinn Hannah , Marshall Austin , Holcomb Morgan , Cruz Leonardo , Soriano Sirena , Treangen Todd J. , Villapol Sonia 
Published: May 2024

AbstractThe gut microbiome is linked to brain pathology in cases of traumatic brain injury (TBI), yet the specific bacteria that are implicated are not well characterized. To address this gap, in this study, we induced traumatic brain injury (TBI) in male C57BL/6J mice using the controlled cortical impact (CCI) injury model. After 35 days, we administered a broad-spectrum antibiotics (ABX) cocktail (ampicillin, gentamicin, metronidazole, vancomycin) through oral gavage for 2 days to diminish existing microbiota. Subsequently, we inflicted a second TBI on the mice and analyzed the neuropathological outcomes five days later. Longitudinal analysis of the microbiome showed significant shifts in the diversity and abundance of bacterial genera during both acute and chronic inflammation. These changes were particularly dramatic following treatment with ABX and after the second TBI. ABX treatment did not affect the production of short-chain fatty acids (SCFA) but did alter intestinal morphology, characterized by reduced villus width and a lower count of goblet cells, suggesting potential negative impacts on intestinal integrity. Nevertheless, diminishing the intestinal microbiome reduced cortical damage, apoptotic cell density, and microglial/macrophage activation in the cortical and thalamic regions of the brain. Our findings suggest that eliminating colonized gut bacteria via broad-spectrum ABX reduces neuroinflammation and enhances neurological outcomes in TBI despite implications to gut health.
Intradental mechano-nociceptors serve as sentinels that prevent tooth damage
Authors: Ronan Elizabeth A. , Gandhi Akash R. , Constantinescu Brian S. C. , Uchima Koecklin Karin H. , Guenther Mak E. , Nagel Maximilian , Blumberg Kaitlyn J. , Stern Tomer , Li Peng , Emrick Joshua J. 
Published: May 2024

ABSTRACTPain is the anticipated output of the trigeminal sensory neurons that innervate the tooth’s vital interior1,2; however, the contribution of intradental neurons to healthy tooth sensation has yet to be defined. Here, we employ in vivo Ca2+imaging to identify and define a population of myelinated high-threshold mechanoreceptors (intradental HTMRs) that detect superficial structural damage of the tooth and initiate jaw opening to protect teeth from damage. Intradental HTMRs remain inactive when direct forces are applied to the intact tooth but become responsive to forces when the structural integrity of the tooth is compromised, and the dentin or pulp is exposed. Their terminals collectively innervate the inner dentin through overlapping receptive fields, allowing them to monitor the superficial structures of the tooth. Indeed, intradental HTMRs detect superficial enamel damage and encode its degree, and their responses persist in the absence of either PIEZO2 or Nav1.83,4. Optogenetic activation of intradental HTMRs triggers a rapid, jaw opening reflex via contraction of the digastric muscle. Taken together, our data indicate that intradental HTMRs serve as sentinels that guard against mechanical threats to the tooth, and their activation results in physical tooth separation to minimize irreversible structural damage. Our work provides a new perspective on the role of intradental neurons as protective rather than exclusively pain-inducing and illustrates additional diversity in the functions of interoreceptors.
Neural mechanisms of resource allocation in working memory
Authors: Li Hsin-Hung , Sprague Thomas C. , Yoo Aspen H. , Ma Wei Ji , Curtis Clayton E. 
Published: May 2024

AbstractTo mitigate capacity limits of working memory, people allocate resources according to an item’s relevance. However, the neural mechanisms supporting such a critical operation remain unknown. Here, we developed computational neuroimaging methods to decode and demix neural responses associated with multiple items in working memory with different priorities. In striate and extrastriate cortex, the gain of neural responses tracked the priority of memoranda. Higher-priority memoranda were decoded with smaller error and lower uncertainty. Moreover, these neural differences predicted behavioral differences in memory prioritization. Remarkably, trialwise variability in the magnitude of delay activity in frontal cortex predicted differences in decoded precision between low and high-priority items in visual cortex. These results suggest a model in which feedback signals broadcast from frontal cortex sculpt the gain of memory representations in visual cortex according to behavioral relevance, thus, identifying a neural mechanism for resource allocation.
Prioritizing working memory resources depends on prefrontal cortex
Authors: Hallenbeck Grace E. , Tardiff Nathan , Sprague Thomas C. , Curtis Clayton E. 
Published: May 2024

ABSTRACTHow the prefrontal cortex contributes to working memory remains controversial, as theories differ in their emphasis on its role in storing memories versus controlling their content. To adjudicate between these competing ideas, we tested how perturbations to the human (both sexes) lateral prefrontal cortex impact the storage and control aspects of working memory during a task that requires human subjects to allocate resources to memory items based on their behavioral priority. Our computational model made a strong prediction that disruption of this control process would counterintuitively improve memory for low-priority items. Remarkably, transcranial magnetic stimulation of retinotopically-defined superior precentral sulcus, but not intraparietal sulcus, unbalanced the prioritization of resources, improving memory for low-priority items as predicted by the model. Therefore, these results provide direct causal support for models in which the prefrontal cortex controls the allocation of resources that support working memory, rather than simply storing the features of memoranda.SIGNIFICANCE STATEMENTAlthough higher-order cognition depends on working memory, the resources that support our memory are severely limited in capacity. To mitigate this limitation, we allocate memory resources according to the behavioral relevance of items. Nonetheless, the neural basis of these abilities remain unclear. Here, we tested the hypothesis that a region in lateral prefrontal cortex controls prioritization in working memory. Indeed, perturbing this region with transcranial magnetic stimulation disrupted the prioritization of working memory resources. Our results provide causal evidence for the hypothesis that prefrontal cortex primarily controls the allocation of memory resources, rather than storing the contents of working memory.
Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting
Authors: Wan Yiming , Helenek Christopher , Coraci Damiano , Balázsi Gábor 
Published: May 2024

SUMMARYThe invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, “collateral damage” by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting fromtranscleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop newMulti-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid(MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.
Active vision in freely moving marmosets using head-mounted eye tracking
Authors: Singh Vikram Pal , Li Jingwen , Dawson Kana , Mitchell Jude F. , Miller Cory T. 
Published: May 2024

AbstractOur understanding of how vision functions as primates actively navigate the real-world is remarkably sparse. As most data have been limited to chaired and typically head-restrained animals, the synergistic interactions of different motor actions/plans inherent to active sensing –e.g. eyes, head, posture, movement, etc.-on visual perception are largely unknown. To address this considerable gap in knowledge, we developed an innovative wireless head-mounted eye tracking system calledCEREBROfor small mammals, such as marmoset monkeys. Our system performsChair-freeEye-Recording usingBackpack mounted micROcontrollers. Because eye illumination and environment lighting change continuously in natural contexts, we developed a segmentation artificial neural network to perform robust pupil tracking in these conditions. Leveraging this innovative system to investigate active vision, we demonstrate that although freely-moving marmosets exhibit frequent compensatory eye movements equivalent to other primates, including humans, the predictability of the visual behavior (gaze) is higher when animals are freely-moving relative to when they are head-fixed. Moreover, despite increases in eye/head-motion during locomotion, gaze stabilization remains steady because of an increase in VOR gain during locomotion. These results demonstrate the efficient, dynamic visuo-motor mechanisms and related behaviors that enable stable, high-resolution foveal vision in primates as they explore the natural world.Significance StatementVision is arguably the most thoroughly understood of all neural systems in the primate brain. Yet there is little known about how vision functions in real-world contexts in which individuals freely move and explore an environment. This dearth in knowledge is largely due to the lack of technology that can accurately track eye-movements in freely-moving individuals with the speed and resolution needed to quantify primate vision. Here we developed an innovative wireless head-mounted eye-tracking system for marmosets that meets these technical needs and enabled us to quantify facts of primate vision in a manner not previously possible, including a set of discoveries that are likely to transform our understanding of this keystone system.
Recombinant and synthetic affibodies function comparably for modulating protein release
Authors: Dorogin Jonathan , Benz Morrhyssey A. , Moore Cameron J. , Benoit Danielle S. W. , Hettiaratchi Marian H. 
Published: May 2024

AbstractPurposeAffibodies are a class of versatile affinity proteins with a wide variety of therapeutic applications, ranging from guided contrast agents for imaging to cell-targeting therapeutics. We have identified several affibodies specific to bone morphogenetic protein-2 (BMP-2) with a range of binding affinities and demonstrated the ability to tune release rate of BMP-2 from affibody-conjugated poly(ethylene glycol) (PEG) hydrogels based on affibody affinity strength. In this work, we compare the purity, structure, and activity of recombinant, bacterially-expressed BMP-2-specific affibodies with affibodies synthesized via solid-phase peptide synthesis.MethodsHigh- and low-affinity BMP-2-specific affibodies were recombinantly expressed using BL21(DE3)E. coliand chemically synthesized using microwave-assisted solid-phase peptide synthesis with Fmoc-Gly-Wang resin. The secondary structures of the affibodies and dissociation constants of affibody-BMP-2 binding were characterized by circular dichroism and biolayer interferometry, respectively. Endotoxin levels were measured using chromogenic limulus amebocyte lysate (LAL) assays. Affibody-conjugated PEG-mal hydrogels were fabricated and loaded with BMP-2 to evaluate hydrogel capacity for controlled release, quantified by enzyme-linked immunosorbent assays (ELISA).ResultsSynthetic and recombinant affibodies were determined to be α-helical by circular dichroism. The synthetic high- and low-affinity BMP-2-specific affibodies demonstrated comparable BMP-2-binding dissociation constants to their recombinant counterparts. Recombinant affibodies retained some endotoxins after purification, while endotoxins were not detected in the synthetic affibodies. High-affinity affibody-conjugated hydrogels reduced cumulative BMP-2 release compared to the low-affinity affibody-conjugated hydrogels and hydrogels without affibodies.ConclusionsSynthetic affibodies demonstrate comparable structure and function to recombinant affibodies, while reducing endotoxin contamination and increasing product yield, indicating that solid-phase peptide synthesis is a viable method of producing affibodies for controlled protein release and other applications.
A novel perspective on equal and cross-frequency neural coupling: integration and segregation of the brain networks’ function
Authors: Mateos Diego M. , Perez Velazquez Jose Luis 
Published: May 2024

AbstractWe introduce a novel perspective in equal and multifrequency coupling derived from considering neuronal synchrony as a possible equivalence relation. The experimental results agree with the theoretical prediction that cross-frequency coupling results in a partition of the brain synchrony state space. We place these results in the framework of the integration and segregation of information in the processing of sensorimotor transformations by the brain cell circuits and propose that equal frequency (1:1) connectivity favours integration of information in the brain whereas cross-frequency coupling (n:m) favours segregation. These observations may provide an outlook about how to reconcile the need for stability in the brain’s operations with the requirement for diversity of activity in order to process many sensorimotor transformations simultaneously.
Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing
Authors: Countryman Andrew D. , Doherty Caroline A. , Herrera-Perez R. Marisol , Kasza Karen E. 
Published: May 2024

AbstractDuring development, epithelia function as malleable substrates that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate the mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in tool expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tagDrosophilaRhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by a stiff basal actomyosin layer. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.
Distinct molecular and functional properties of human induced-proprioceptor and low-threshold mechanoreceptor neurons
Authors: Hulme Amy J. , Finol-Urdaneta Rocio K. , McArthur Jeffrey R. , Marzano Nicholas R. , Maksour Simon , Thind Amarinder , Guo Yang , Kaul Dominic , Maddock Marnie , Friedrich Oliver , Martinac Boris , Adams David J. , Dottori Mirella 
Published: May 2024

AbstractSensing mechanical stimuli is crucial for the function of internal and external tissues, such as the skin and muscles. Much of our understanding of mechanosensory physiology relies on rodent studies, which may not directly translate to humans. To address the knowledge gap in human mechanosensation, we developed distinct populations of human mechanosensory neuronal subtypes from human pluripotent stem cells (hPSC). By inducing co-expression of NGN2/RUNX3 or NGN2/SHOX2 in hPSC-derived migrating neural crest cells we directed their specification to proprioceptor and low-threshold mechanoreceptor neuronal subtypes, respectively. The induced neurons exhibited transcriptional profiles consistent with mechanosensory neurons and displayed functional responses to mechanical stimuli, such as stretch and submicrometer probe indentation to the soma. Notably, each subtype displayed unique mechanical thresholds and desensitization properties akin to proprioceptors and low-threshold mechanoreceptors and both induced neuronal subtypes fired action potentials in response to minute mechanical stimuli, predominantly relying on PIEZO2 for mechanosensory function. Collectively, this study provides a foundational model for exploring human neuronal mechanosensory biology.
Non-canonical function of ADAM10 in presynaptic plasticity
Authors: Bär Julia , Fanutza Tomas , Reimann Christopher C. , Seipold Lisa , Grohe Maja , Bolter Janike Rabea , Delfs Flemming , Bucher Michael , Gee Christine E , Schweizer Michaela , Saftig Paul , Mikhaylova Marina 
Published: May 2024

AbstractA Disintegrin And Metalloproteinase 10 (ADAM10) plays a pivotal role in shaping neuronal networks by orchestrating the activity of numerous membrane proteins through the shedding of their extracellular domains. Despite its significance in the brain, the specific cellular localization of ADAM10 remains not well understood due to a lack of appropriate tools. Here, using a specific ADAM10 antibody suitable for immunostainings, we discover that ADAM10 is localized to presynapses and especially enriched at presynaptic vesicles of mossy fiber (MF)-CA3 synapses in the hippocampus. These synapses undergo pronounced frequency facilitation of neurotransmitter release, a process that play critical roles in information transfer and neural computation. We demonstrate, that in conditional ADAM10 knockout mice the ability of MF synapses to undergo this type of synaptic plasticity is greatly reduced. The loss of facilitation depends on the cytosolic domain of ADAM10 and association with the calcium sensor synaptotagmin 7 rather than its proteolytic activity. Our findings unveil a new pathway contributing to the regulation of synaptic vesicle exocytosis.
PTPRS is a novel marker for early tau pathology and synaptic integrity in Alzheimer’s disease
Authors: Poirier Alexandre , Picard Cynthia , Labonté Anne , Aubry Isabelle , Auld Daniel , Zetterberg Henrik , Blennow Kaj ,   the PREVENT-AD research group, Tremblay Michel L. , Poirier Judes 
Published: May 2024

AbstractWe examined the role of protein tyrosine phosphatase receptor sigma (PTPRS) in the context of Alzheimer’s disease and synaptic integrity. Publicly available datasets (BRAINEAC, ROSMAP, ADC1) and a cohort of asymptomatic but “at risk” individuals (PREVENT-AD) were used to explore the relationship between PTPRS and various Alzheimer’s disease biomarkers. We identified that PTPRS rs10415488 variant C shows features of neuroprotection against early tau pathology and synaptic degeneration in Alzheimer’s disease. This single nucleotide polymorphism correlated with higher PTPRS transcript abundance and lower P-tau181 and GAP-43 levels in the CSF. In the brain, PTPRS protein abundance was significantly correlated with the quantity of two markers of synaptic integrity: SNAP25 and SYT-1. We also found the presence of sexual dimorphism for PTPRS, with higher CSF concentrations in males than females. Male carriers for variant C were found to have a 10-month delay in the onset of AD. We thus conclude that PTPRS acts as a neuroprotective receptor in Alzheimer’s disease. Its protective effect is most important in males, in whom it postpones the age of onset of the disease.