bioRxiv : the preprint server for biology最新文献

The asexual stages of Toxoplasma gondii are defined by the rapidly growing tachyzoite during the acute infection and by the slow growing bradyzoite housed within tissue cysts during the chronic infection. These stages represent unique physiological states, each with distinct glucans reflecting differing metabolic needs. A defining feature of T. gondii bradyzoites is the presence of insoluble storage glucans known as amylopectin granules (AGs) that are believed to play a role in reactivation, but their functions during the chronic infection remain largely unexplored. More recently, the presence of storage glucans has been recognized in tachyzoites where their precise function and architecture have yet to be fully defined. Importantly, the T. gondii genome encodes activities needed for glucan turnover: a glucan phosphatase (TgLaforin; TGME49_205290) and a glucan kinase (TgGWD; TGME49_214260) that catalyze a cycle of reversible glucan phosphorylation required for glucan degradation by amylases. The expression of these enzymes in tachyzoites supports the existence of a storage glucan, evidence that is corroborated by specific labeling with the anti-glycogen antibody IV58B6. Disruption of reversible glucan phosphorylation via a CRISPR/Cas9 knockout (KO) of TgLaforin revealed no growth defects under nutrient-replete conditions in tachyzoites. However, the growth of TgLaforin-KO tachyzoites was severely stunted when starved of glutamine, even under glucose replete conditions. The loss of TgLaforin also resulted in the attenuation of acute virulence in mice accompanied by a lower cyst burden. While the absence of TgLaforin did not impact the size distribution of tissue cysts, bradyzoites within both in vitro generated and in vivo tissue cysts exhibited profound changes in AG levels and morphology. Quantification of relative AG levels using AmyloQuant, an imaging-based application, revealed the starch-excess phenotype associated with the loss of TgLaforin is heterogeneous across tissue cysts and exhibits a temporal profile linked to an emerging AG cycle. Together, these data demonstrate the importance of glucan turnover across the T. gondii asexual cycle. These findings, alongside our previously identified class of small molecules that inhibit TgLaforin, implicate reversible glucan phosphorylation as a legitimate target for the development of new drugs against chronic T. gondii infections.

Background: Modeling brain stimulation at the microscopic scale may reveal new paradigms for a variety of stimulation modalities.

Objective: We present the largest map of distributions of the extracellular electric field to date within a layer L2/L3 mouse primary visual cortex brain sample, which was enabled by automated analysis of serial section electron microscopy images with improved handling of image defects (250×140×90 μm ³ volume).

Methods: We used the map to identify microscopic perturbations of the extracellular electric field and their effect on the activating thresholds of individual neurons. Previous relevant studies modeled a macroscopically homogeneous cortical volume. Result: Our immediate result is a reduction of the predicted stimulation field strength necessary for neuronal activation by a factor of approximately 0.7 (or by 30%) on average, due to microscopic perturbations of the extracellular electric field-an electric field "spatial noise" with a mean value of zero.

Conclusion: Although this result is largely sample-specific, it aligns with experimental data indicating that existing macroscopic theories substantially overestimate the electric fields necessary for brain stimulation.

Significance statement: Currently, there is a discrepancy between macroscopic volumetric brain modeling for brain stimulation and experimental results: experiments typically reveal lower electric intensities required for brain stimulation. This study is arguably the first attempt to model brain stimulation at the microscopic scale, enabled by automated analysis of modern scanning electron microscopy images of the brain. The immediate result is a prediction of lower electric field intensities necessary for brain stimulation, with an average reduction factor of 0.7.

Both enhanced motion-induced nausea and increased static imbalance are observed symptoms in migraine and especially vestibular migraine (VM). Motion-induced nausea and static imbalance were investigated in a mouse model, nestin/hRAMP1, expressing elevated levels of human RAMP1 which enhances CGRP signaling in the nervous system, and compared to non-affected littermate controls. Behavioral surrogates such as the motion- induced thermoregulation and postural sway center of pressure (CoP) assays were used to assess motion sensitivity. Nausea readouts revealed that the nestin/hRAMP1 mouse exhibit an increased sensitivity to CGRP's effects at lower doses compared to unaffected controls. In addition, the nestin/hRAMP1 mice exhibit a higher dynamic range in postural sway than their wildtype counterparts, along with increased sway observed in nestin/hRAMP1 male mice that was not present in male unaffected controls. Results from migraine blocker experiments were challenging to interpret, but the data suggests that olcegepant is incapable of reversing CGRP-induced or endogenous alterations in the nestin/hRAMP1 mice, while rizatriptan was ineffective in both the nestin/hRAMP1 and control mice. The results indicate that overexpression of hRAMP1 leads to heightened endogenous CGRP signaling. Results also suggest that both olcegepant and rizatriptan are ineffective in reducing nausea and sway in this hypersensitive CGRP mouse model. This study suggests that the hypersensitive nestin/hRAMP1 mouse may serve as a model for difficult to treat cases of migraine that exhibit increased motion sensitivity.

Sleep disturbances are associated with poor long-term memory (LTM) formation, yet the underlying cell types and neural circuits involved have not been fully decoded. Dopamine neurons (DANs) are involved in memory processing at multiple stages. Here, using both male and female flies, Drosophila melanogaster , we show that, during the first few hours of memory consolidation, disruption of basal activity of a small subset of protocerebral anterior medial DANs (PAM-DANs), by either brief activation or inhibition of the two dorsal posterior medial (DPM) neurons, impairs 24 h LTM. Interestingly, these brief changes in activity using female flies result in sleep loss and fragmentation, especially at night. Pharmacological rescue of sleep after manipulation restores LTM. A specific subset of PAM-DANs (PAM-α1) that synapse onto DPM neurons specify the microcircuit that links sleep and memory. PAM-DANs, including PAM-α1, form functional synapses onto DPM mainly via multiple dopamine receptor subtypes. This PAM-α1 to DPM microcircuit exhibits a synchronized, transient, post-training increase in activity during the critical memory consolidation window, suggesting an effect of this microcircuit on maintaining the sleep necessary for LTM consolidation. Our results provide a new cellular and circuit basis for the complex relationship between sleep and memory.

There is a significant need for scalable CRISPR-based genetic screening methods that can be applied directly in mammalian tissues in vivo while enabling cell type-specific analysis. To address this, we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in the mouse tissues. We demonstrate the utility of this approach by screening two distinct large sgRNA libraries, together targeting over 5,000 genes, in mouse brains to create a robust profile of neuron-essential genes. We validate two genes as strongly neuron-essential in both primary mouse neurons and in vivo , confirming the predictive power of our platform. By comparing results from individual mice and across different cell populations, we highlight the reproducibility and scalability of the platform and show that it is highly sensitive even for screening smaller neuronal subpopulations. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and multiplicity of infection on screen performance to establish general guidelines for large-scale in vivo screens.

In addition to its role as cellular energy currency, adenosine triphosphate (ATP) serves as an extracellular messenger that mediates diverse cell-to-cell communication. Compelling evidence supports that ATP is released from cells through pannexins, a family of membrane proteins that form heptameric large-pore channels. However, the activation mechanisms that trigger ATP release by pannexins remain poorly understood. Here, we discover lysophospholipids as endogenous pannexin activators, using activity-guided fractionation of mouse tissue extracts combined with untargeted metabolomics and electrophysiology. We show that lysophospholipids directly and reversibly activate pannexins in the absence of other proteins. Secretomics experiments reveal that lysophospholipid-activated pannexin 1 leads to the release of not only ATP but also other signaling metabolites, such as 5'-methylthioadenosine, which is important for immunomodulation. We also demonstrate that lysophospholipids activate endogenous pannexin 1 in human monocytes, leading to the release of IL-1β through inflammasome activation. Our results provide a connection between lipid metabolism and purinergic signaling, both of which play major roles in immune responses.

The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. Here, we show that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in early pre-malignant cells when they overproliferate among wildtype cells, in healthy cells when they later eliminate pre-malignant cells, and by supercompetitors as they compete to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. We demonstrate that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well-defined functions of Fmi.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that utilizes its type III secretion systems (T3SSs) to inject virulence factors into host cells and colonize the host. In turn, a subset of cytosolic immune receptors respond to T3SS ligands by forming multimeric signaling complexes called inflammasomes, which activate caspases that induce interleukin-1 (IL-1) family cytokine release and an inflammatory form of cell death called pyroptosis. Human macrophages mount a multifaceted inflammasome response to Salmonella infection that ultimately restricts intracellular bacterial replication. However, how inflammasomes restrict Salmonella replication remains unknown. We find that caspase-1 is essential for mediating inflammasome responses to Salmonella and restricting bacterial replication within human macrophages, with caspase-4 contributing as well. We also demonstrate that the downstream pore-forming protein gasdermin D (GSDMD) and Ninjurin-1 (NINJ1), a mediator of terminal cell lysis, play a role in controlling Salmonella replication in human macrophages. Notably, in the absence of inflammasome responses, we observed hyperreplication of Salmonella within the cytosol of infected cells as well as increased bacterial replication within vacuoles, suggesting that inflammasomes control Salmonella replication primarily within the cytosol and also within vacuoles. These findings reveal that inflammatory caspases and pyroptotic factors mediate inflammasome responses that restrict the subcellular localization of intracellular Salmonella replication within human macrophages.

Producing dense 3D reconstructions from biological imaging data is a challenging instance segmentation task that requires significant ground-truth training data for effective and accurate deep learning-based models. Generating training data requires intense human effort to annotate each instance of an object across serial section images. Our focus is on the especially complicated brain neuropil, comprising an extensive interdigitation of dendritic, axonal, and glial processes visualized through serial section electron microscopy. We developed a novel deep learning-based method to generate dense 3D segmentations rapidly from sparse 2D annotations of a few objects on single sections. Models trained on the rapidly generated segmentations achieved similar accuracy as those trained on expert dense ground-truth annotations. Human time to generate annotations was reduced by three orders of magnitude and could be produced by non-expert annotators. This capability will democratize generation of training data for large image volumes needed to achieve brain circuits and measures of circuit strengths.

The lung's mechanosensitive immune response, which occurs when pulmonary alveoli are overstretched, is a major impediment to ventilation therapy for hypoxemic respiratory failure. The cause is not known. We tested the hypothesis that alveolar stretch causes stretch of alveolar macrophages (AMs), leading to the immune response. In lungs viewed by optical imaging, sessile AMs expressed gap junctional protein connexin-43 (Cx43), and they communicated with the alveolar epithelium through gap junctions. Alveolar hyperinflation increased Ca ²⁺ in the AMs but did not stretch the AMs. The Ca ²⁺ response, and concomitant TNFα secretion by AMs were blocked in mice with AM-specific deletion of Cx43. The AM responses, as also lung injury due to mechanical ventilation at high tidal volume, were inhibited by AM-specific delivery of lipid nanoparticles containing Xestospongin C, which blocked the induced Ca ²⁺ increases. We conclude, Cx43- and Ca ²⁺ -dependent AM-epithelial interactions determine the lung's mechanosensitive immunity, providing a basis for therapy for ventilator- induced lung injury.