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PLOS Biology: New Articles

  • Highly accurate image registration for 3D multiplexed cyclic imaging using dense labeling in expandable tissue gels

    by Hyunwoo Kim, Joon-Goon Kim, Jueun Sim, Hoyeon Nam, In Cho, Hyejin Shin, Junyoung Kwon, Dae-Hyeon Song, Seoungbin Bae, Young-Gyu Yoon, Taeyun Ku, Jae-Byum Chang

    Multiplexed cyclic imaging in expandable tissue gels has been extensively studied to visualize numerous biomolecules at a nanoscale resolution in situ. Previous studies have employed sparse labels, such as DAPI or lectin staining, as registration markers. However, these sparse labels do not adequately capture the full extent of deformation across the entire region of interest. To overcome this challenge, we propose the use of dense labels, specifically fluorophore N-hydroxysuccinimide (NHS)-ester staining, as registration markers to achieve highly accurate image registration. We first tested several NHS-functionalized fluorophores as fiducial markers and identified the proper candidates for three-dimensional (3D) multiplexed cyclic imaging. We analyzed the registration accuracy between DAPI and NHS-ester staining and illustrated that dense label-based registration provides a more accurate registration performance. In the multiplexed imaging of expanded specimens, we observed that repetitive expansion/shrinking processes and chemical treatments for signal elimination can induce 3D nonlinear distortion. This sample distortion can be mitigated by re-embedding the tissue gel or replacing the chemical de-staining process with photobleaching-based signal removal or computational signal unmixing. With such an optimized experimental setup, we demonstrated 3D multiplexed cyclic imaging with nanoscale precision image registration. Finally, we prove that dense biological structures, such as actin, can be used as registration markers to achieve high registration accuracy. We anticipate that the proposed dense labeling strategy will overcome the technical limitations of multiplexed cyclic imaging in expandable tissue gels, offering high-precision registration. We expect it to be widely adopted by the biological and medical communities.

  • Group B <i>Streptococci</i> lyse endothelial cells to infect the brain in a zebrafish meningitis model

    by Sumedha Ravishankar, Samantha M. Tuohey, Nicole O. Ramos, Satoshi Uchiyama, Megan I. Hayes, Kalisa Kang, Victor Nizet, Cressida A. Madigan

    To cause meningitis, bacteria move from the bloodstream to the brain, crossing the endothelial cells of the blood–brain barrier. Most studies on how bacteria cross the blood–brain barrier have been performed in vitro using cultured endothelial cells, due to a paucity of animal models. Group B Streptococcus (GBS) is the leading cause of bacterial meningitis in neonates and is primarily thought to cross the blood–brain barrier by transcytosis through endothelial cells. To test this hypothesis in vivo, we used optically transparent zebrafish larvae. Time-lapse confocal microscopy revealed that GBS forms extracellular microcolonies in brain blood vessels and causes perforation and lysis of blood–brain barrier endothelial cells, which promotes bacterial entry into the brain. Vessels infected with GBS microcolonies were distorted and contained thrombi. Inhibition of clotting worsened brain invasion, suggesting a host-protective role for thrombi. The GBS lysin cylE, implicated in brain invasion in vitro, was found dispensable in vivo. Instead, pro-inflammatory mediators associated with endothelial cell damage and blood–brain barrier breakdown were specifically upregulated in the zebrafish head upon GBS entry into the brain. Therefore, GBS crosses the blood–brain barrier in vivo not by transcytosis, but by endothelial cell lysis and death. Given that we observe the same invasion route for a meningitis-associated strain of Streptococcus pneumoniae, our findings suggest that streptococcal infection of brain blood vessels triggers endothelial cell inflammation and lysis, thereby facilitating brain invasion.

  • Plasma membrane remodeling in GM2 gangliosidoses drives synaptic dysfunction

    by Alex S. Nicholson, David A. Priestman, Robin Antrobus, James C. Williamson, Reuben Bush, Shannon J. McKie, Henry G. Barrow, Emily Smith, Kostantin Dobrenis, Nicholas A. Bright, Frances M. Platt, Janet E. Deane

    Glycosphingolipids (GSL) are important bioactive membrane components. GSLs containing sialic acids, known as gangliosides, are highly abundant in the brain and diseases of ganglioside metabolism cause severe early-onset neurodegeneration. The ganglioside GM2 is processed by β-hexosaminidase A and when non-functional GM2 accumulates causing Tay–Sachs and Sandhoff diseases. We have developed i3Neuron-based disease models demonstrating storage of GM2 and severe endolysosomal dysfunction. Additionally, the plasma membrane (PM) is significantly altered in its lipid and protein composition. These changes are driven in part by lysosomal exocytosis causing inappropriate accumulation of lysosomal proteins on the cell surface. There are also significant changes in synaptic protein abundances with direct functional impact on neuronal activity. Lysosomal proteins are also enriched at the PM in GM1 gangliosidosis supporting that lysosomal exocytosis is a conserved mechanism of PM proteome change in these diseases. This work provides mechanistic insights into neuronal dysfunction in gangliosidoses highlighting that these are severe PM disorders with implications for other lysosomal and neurodegenerative diseases.

  • Cuticular collagens mediate cross-kingdom predator–prey interactions between trapping fungi and nematodes

    by Han-Wen Chang, Hung-Che Lin, Ching-Ting Yang, Rebecca J. Tay, Dao-Ming Chang, Yi-Chung Tung, Yen-Ping Hsueh

    Adhesive interactions, mediated by specific molecular and structural mechanisms, are fundamental to host–pathogen and predator–prey relationships, driving evolutionary dynamics and ecological interactions. Here, we investigate the cellular and molecular basis of adhesion between the nematode Caenorhabditis elegans and its natural predator the nematode-trapping fungus Arthrobotrys oligospora, which employs specialized adhesive nets to capture its prey. Using forward genetic screens, we identified C. elegans mutants that escape fungal traps and revealed the nuclear hormone receptor NHR-66 as a key regulator of fungal-nematode adhesion. Loss-of-function mutations in nhr-66 conferred resistance to fungal trapping through the downregulation of a large subset of cuticular collagen genes. Restoring collagen gene expression in nhr-66 mutants abolished the escape phenotype, highlighting the essential role of these structural proteins in fungal-nematode adhesion. Furthermore, sequence analysis of natural C. elegans populations revealed no obvious loss-of-function variants in nhr-66, suggesting selective pressures exist that balance adhesion-mediated predation risk with physiological robustness. We observed that loss of nhr-66 function resulted in a trade-off of increased hypersensitivity to hypoosmotic stress and cuticular fragility. These findings underscore the pivotal role of structural proteins in shaping ecological interactions and the evolutionary arms race between predator and prey.

  • Functional connectivity and GABAergic signaling modulate the enhancement effect of neurostimulation on mathematical learning

    by George Zacharopoulos, Masoumeh Dehghani, Beatrix Krause-Sorio, Jamie Near, Roi Cohen Kadosh

    Effortful learning and practice are integral to academic attainment in areas like reading, language, and mathematics, shaping future career prospects, socioeconomic status, and health outcomes. However, academic learning outcomes often exhibit disparities, with initial cognitive advantages leading to further advantages (the Matthew effect). One of the areas in which learners frequently exhibit difficulties is mathematical learning. Neurobiological research has underscored the involvement of the dorsolateral prefrontal cortex (dlPFC), the posterior parietal cortex (PPC), and the hippocampus in mathematical learning. However, their causal contributions remain unclear. Moreover, recent findings have highlighted the potential role of excitation/inhibition (E/I) balance in neuroplasticity and learning. To deepen our understanding of the mechanisms driving mathematical learning, we employed a novel approach integrating double-blind excitatory neurostimulation—high-frequency transcranial random noise stimulation (tRNS)—and examined its effect at the behavioral, functional, and neurochemical levels. During a 5-day mathematical learning paradigm (n = 72) active tRNS was applied over the dlPFC or the PPC, and we compared the effects versus sham tRNS. Individuals exhibiting stronger positive baseline frontoparietal connectivity demonstrated greater improvement in calculation learning. Subsequently, utilizing tRNS to modulate frontoparietal connectivity, we found that participants with weaker positive baseline frontoparietal connectivity, typically associated with poorer learning performance, experienced enhanced learning outcomes following dlPFC-tRNS only. Further analyses revealed that dlPFC-tRNS improved learning outcomes for participants who showed reductions in dlPFC GABA when it was accompanied by a reduced positive frontoparietal connectivity, but this effect was reversed for participants who showed increased positive frontoparietal connectivity. Our multimodal approach elucidates the causal role of the dlPFC and frontoparietal network in a critical academic learning skill, shedding light on the interplay between functional connectivity and GABAergic modulation in the efficacy of brain-based interventions to augment learning outcomes, particularly benefiting individuals who would learn less optimally based on their neurobiological profile.