Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607203
Mariona Fucho-Rius, Smitha Maretvadakethope, R. Pérez-Carrasco, Àlex Haro, Tomás Alarcón, J. Sardanyés
The behavior of nonlinear systems close to critical transitions has relevant implications in assessing complex systems’ stability, transient properties, and resilience. Transient times become extremely long near phase transitions (or bifurcations) in a phenomenon generically known as critical slowing down, observed in electronic circuits, quantum electrodynamics, ferromagnetic materials, ecosystems, and gene regulatory networks. Typically, these transients follow well-defined universal laws of the form τ ∼ |µ − µc| β, describing how their duration, τ, varies as the control parameter, µ, approaches its critical value, µc. For instance, transients’ delays right after a saddle-node (SN) bifurcation, influenced by so-called ghosts, follow β = −1/2. Despite intensive research on slowing down phenomena over the past decades for single bifurcations, both local and global, the behavior of transients when several bifurcations are close to each other remains unknown. Here, we study transients close to two SN bifurcations collapsing into a transcritical one. To do so, we analyze a simple nonlinear model of a self-activating gene regulated by an external signal that exhibits a mushroom bifurcation. We also propose and study a normal form for a system with two SN bifurcations merging into a transcritical one. For both systems, we show analytical and numerical evidence of a synergistic increase in transients due to the coupling of the two ghosts and the transcritical slowing down. We also explore the influence of noise on the transients in the gene-regulatory model. We show that intrinsic and extrinsic noise play opposite roles in the slowing down of the transition allowing us to control the timing of the transition without compromising the precision of the timing. This establishes novel molecular strategies to generate genetic timers with transients much larger than the typical timescales of the reactions involved.
{"title":"Local Nearby Bifurcations Lead to Synergies in Critical Slowing Down: the Case of Mushroom Bifurcations","authors":"Mariona Fucho-Rius, Smitha Maretvadakethope, R. Pérez-Carrasco, Àlex Haro, Tomás Alarcón, J. Sardanyés","doi":"10.1101/2024.08.08.607203","DOIUrl":"https://doi.org/10.1101/2024.08.08.607203","url":null,"abstract":"The behavior of nonlinear systems close to critical transitions has relevant implications in assessing complex systems’ stability, transient properties, and resilience. Transient times become extremely long near phase transitions (or bifurcations) in a phenomenon generically known as critical slowing down, observed in electronic circuits, quantum electrodynamics, ferromagnetic materials, ecosystems, and gene regulatory networks. Typically, these transients follow well-defined universal laws of the form τ ∼ |µ − µc| β, describing how their duration, τ, varies as the control parameter, µ, approaches its critical value, µc. For instance, transients’ delays right after a saddle-node (SN) bifurcation, influenced by so-called ghosts, follow β = −1/2. Despite intensive research on slowing down phenomena over the past decades for single bifurcations, both local and global, the behavior of transients when several bifurcations are close to each other remains unknown. Here, we study transients close to two SN bifurcations collapsing into a transcritical one. To do so, we analyze a simple nonlinear model of a self-activating gene regulated by an external signal that exhibits a mushroom bifurcation. We also propose and study a normal form for a system with two SN bifurcations merging into a transcritical one. For both systems, we show analytical and numerical evidence of a synergistic increase in transients due to the coupling of the two ghosts and the transcritical slowing down. We also explore the influence of noise on the transients in the gene-regulatory model. We show that intrinsic and extrinsic noise play opposite roles in the slowing down of the transition allowing us to control the timing of the transition without compromising the precision of the timing. This establishes novel molecular strategies to generate genetic timers with transients much larger than the typical timescales of the reactions involved.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141923194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.607055
Timothy J. Duerr, Melissa Miller, Sage Kumar, Dareen Bakr, Jackson R. Griffiths, Aditya K. Gautham, Danielle Douglas, S. R. Voss, James R. Monaghan
Regenerating limbs retain their proximodistal (PD) positional identity following amputation. This positional identity is genetically encoded by PD patterning genes that instruct blastema cells to regenerate the appropriate PD limb segment. Retinoic acid (RA) is known to specify proximal limb identity, but how RA signaling levels are established in the blastema is unknown. Here, we show that RA breakdown via CYP26B1 is essential for determining RA signaling levels within blastemas. CYP26B1 inhibition molecularly reprograms distal blastemas into a more proximal identity, phenocopying the effects of administering excess RA. We identify Shox as an RA-responsive gene that is differentially expressed between proximally and distally amputated limbs. Ablation of Shox results in shortened limbs with proximal skeletal elements that fail to initiate endochondral ossification. These results suggest that PD positional identity is determined by RA degradation and RA-responsive genes that regulate PD skeletal element formation during limb regeneration.
肢体截肢后，再生肢体会保留其近端（PD）位置特征。这种位置特征由PD模式基因遗传编码，这些基因指示胚泡细胞再生适当的PD肢段。已知视黄酸（RA）可指定肢体近端特征，但视黄酸信号水平如何在胚泡中建立尚不清楚。在这里，我们发现通过 CYP26B1 分解 RA 对确定胚泡内的 RA 信号水平至关重要。抑制 CYP26B1 可在分子上将远端胚泡重新编程为更近端特征，从而表征了过量 RA 的影响。我们发现 Shox 是一种 RA 响应基因，它在近端和远端截肢肢体中表达不同。消减 Shox 会导致肢体缩短，其近端骨骼元素无法启动软骨内骨化。这些结果表明，在肢体再生过程中，PD的位置特性是由RA降解和RA反应基因决定的，而RA反应基因可调控PD骨骼元件的形成。
{"title":"Retinoic acid breakdown is required for proximodistal positional identity during amphibian limb regeneration","authors":"Timothy J. Duerr, Melissa Miller, Sage Kumar, Dareen Bakr, Jackson R. Griffiths, Aditya K. Gautham, Danielle Douglas, S. R. Voss, James R. Monaghan","doi":"10.1101/2024.08.07.607055","DOIUrl":"https://doi.org/10.1101/2024.08.07.607055","url":null,"abstract":"Regenerating limbs retain their proximodistal (PD) positional identity following amputation. This positional identity is genetically encoded by PD patterning genes that instruct blastema cells to regenerate the appropriate PD limb segment. Retinoic acid (RA) is known to specify proximal limb identity, but how RA signaling levels are established in the blastema is unknown. Here, we show that RA breakdown via CYP26B1 is essential for determining RA signaling levels within blastemas. CYP26B1 inhibition molecularly reprograms distal blastemas into a more proximal identity, phenocopying the effects of administering excess RA. We identify Shox as an RA-responsive gene that is differentially expressed between proximally and distally amputated limbs. Ablation of Shox results in shortened limbs with proximal skeletal elements that fail to initiate endochondral ossification. These results suggest that PD positional identity is determined by RA degradation and RA-responsive genes that regulate PD skeletal element formation during limb regeneration.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141923970","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607139
Kevin Jayaraj, Ritesh Kumar, Sukanya Shyamasundar, Jai S. Polepalli, T. Arumugam, S. T. Dheen
Ischemic stroke significantly contributes to global morbidity and disability through a cascade of neurological responses. Microglia, the immune modulators within the brain, exhibit dual roles in exacerbating and ameliorating ischemic injury through neuroinflammatory and neuroprotective roles, respectively. Despite emerging insights into microglia’s role in neuronal support, the potential of epigenetic intervention to modulate microglial activity remains largely unexplored. We have previously shown that sodium butyrate, a histone deacetylase inhibitor (HDACi) epigenetically regulates inflammatory response of microglia after ischemic stroke and this study was aimed to characterize the transcriptomic profiles of microglia and their spatial distribution in the stroke brain followed by HDACi administration. We hypothesized that the administration of HDACi epigenetically modulates microglial activation and a region-specific microglial phenotype in the stroke brain, shifting their phenotype from neurotoxic to neuroprotective and facilitating neuronal repair and recovery in the ischemic penumbra. Utilizing a rodent model of middle cerebral artery occlusion (MCAo), spatial transcriptomics and 3D morphometric reconstruction techniques were employed to investigate microglial responses in critical penumbral regions, such as the hippocampus, thalamus, cortex and striatum following HDACi administration. We found that HDACi significantly altered the microglial transcriptomic landscape involving biological pathways of neuroinflammation, neuroprotection and phagocytosis as well as morphological phenotype, promoting a shift towards reparative, neurotrophic profiles within the ischemic penumbra. These changes were also associated with enhanced neuronal survival and reduced neuroinflammation in specific regions in the ischemic brain. By elucidating the mechanisms through which HDAC inhibition influences microglial function, our findings propose therapeutic avenues for neuroprotection and rehabilitation in ischemic stroke, and possibly other neurodegenerative conditions that involve microglia-mediated neuroinflammation.
{"title":"Spatial Transcriptomic Analysis Reveals HDAC Inhibition Modulates Microglial Dynamics to Protect Against Ischemic Stroke in Mice","authors":"Kevin Jayaraj, Ritesh Kumar, Sukanya Shyamasundar, Jai S. Polepalli, T. Arumugam, S. T. Dheen","doi":"10.1101/2024.08.08.607139","DOIUrl":"https://doi.org/10.1101/2024.08.08.607139","url":null,"abstract":"Ischemic stroke significantly contributes to global morbidity and disability through a cascade of neurological responses. Microglia, the immune modulators within the brain, exhibit dual roles in exacerbating and ameliorating ischemic injury through neuroinflammatory and neuroprotective roles, respectively. Despite emerging insights into microglia’s role in neuronal support, the potential of epigenetic intervention to modulate microglial activity remains largely unexplored. We have previously shown that sodium butyrate, a histone deacetylase inhibitor (HDACi) epigenetically regulates inflammatory response of microglia after ischemic stroke and this study was aimed to characterize the transcriptomic profiles of microglia and their spatial distribution in the stroke brain followed by HDACi administration. We hypothesized that the administration of HDACi epigenetically modulates microglial activation and a region-specific microglial phenotype in the stroke brain, shifting their phenotype from neurotoxic to neuroprotective and facilitating neuronal repair and recovery in the ischemic penumbra. Utilizing a rodent model of middle cerebral artery occlusion (MCAo), spatial transcriptomics and 3D morphometric reconstruction techniques were employed to investigate microglial responses in critical penumbral regions, such as the hippocampus, thalamus, cortex and striatum following HDACi administration. We found that HDACi significantly altered the microglial transcriptomic landscape involving biological pathways of neuroinflammation, neuroprotection and phagocytosis as well as morphological phenotype, promoting a shift towards reparative, neurotrophic profiles within the ischemic penumbra. These changes were also associated with enhanced neuronal survival and reduced neuroinflammation in specific regions in the ischemic brain. By elucidating the mechanisms through which HDAC inhibition influences microglial function, our findings propose therapeutic avenues for neuroprotection and rehabilitation in ischemic stroke, and possibly other neurodegenerative conditions that involve microglia-mediated neuroinflammation.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141923856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607230
Giriraj Sahu, Dylan Greening, Wilten Nicola, Ray W. Turner
Sodium and potassium channels that regulate axonal spike propagation are highly organized at nodes of Ranvier by a spectrin-actin membrane periodic skeleton. STORM-TIRF microscopy was used to define the spatial organization over the soma of a complex of Cav1.3 calcium, RyR2, and IK potassium channels (CaRyK complex) that generate a slow AHP in hippocampal neurons. Nearest neighbor distance and non-negative matrix factorization analyses identified two spatial patterns as linear rows of 3-8 immuno-labeled clusters with 155 nm periodicity that extended to branchpoints, or as isolated clusters with 600-800 nm separation. The rows and isolated clusters for each of the CaRyK complex proteins closely overlapped with the patterns for spectrin βII and the actin linking proteins actinin I and II. Together the data reveal a close correspondence between the placement of CaRyK complex proteins and that of a net-like organization of spectrin βII across the soma. The regularity in the pattern of expression of these proteins at ER-PM junctions suggest their role as functional nodes of calcium- and calcium-gated potassium channels to control the pattern of spike output at the soma.
{"title":"Ion channels that mediate calcium-dependent control of spike patterns are spatially organized across the soma in relation to a cytoskeletal assembly","authors":"Giriraj Sahu, Dylan Greening, Wilten Nicola, Ray W. Turner","doi":"10.1101/2024.08.08.607230","DOIUrl":"https://doi.org/10.1101/2024.08.08.607230","url":null,"abstract":"Sodium and potassium channels that regulate axonal spike propagation are highly organized at nodes of Ranvier by a spectrin-actin membrane periodic skeleton. STORM-TIRF microscopy was used to define the spatial organization over the soma of a complex of Cav1.3 calcium, RyR2, and IK potassium channels (CaRyK complex) that generate a slow AHP in hippocampal neurons. Nearest neighbor distance and non-negative matrix factorization analyses identified two spatial patterns as linear rows of 3-8 immuno-labeled clusters with 155 nm periodicity that extended to branchpoints, or as isolated clusters with 600-800 nm separation. The rows and isolated clusters for each of the CaRyK complex proteins closely overlapped with the patterns for spectrin βII and the actin linking proteins actinin I and II. Together the data reveal a close correspondence between the placement of CaRyK complex proteins and that of a net-like organization of spectrin βII across the soma. The regularity in the pattern of expression of these proteins at ER-PM junctions suggest their role as functional nodes of calcium- and calcium-gated potassium channels to control the pattern of spike output at the soma.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141923991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.607090
Sara Ghanbarpour Mamaghani, Joanna B. Dahl
The micromechanical measurement field has struggled to establish repeatable techniques, likely because the deforming stresses can be complicated and difficult to model. Here we demonstrate experimentally the ability of cross-slot microfluidic device to create a quasi-steady deformation state in agarose hydrogel microparticles to replicate a traditional uniaxial creep test at the microscale and at relatively high throughput. A recent numerical study by Lu et al. [Lu, Guo, Yu, Sui. J. Fluid Mech., 2023, 962, A26] showed that viscoelastic capsules flowing through a cross-slot can achieve a quasi-steady strain near the extensional flow stagnation point that is equal to the equilibrium static strain, thereby implying that continuous operation of a cross-slot can accurately capture capsule elastic mechanical behavior in addition to transient behavior. However, no microfluidic cross-slot studies have reported quasi-steady strains for suspended cells or particles, to our knowledge. By using large dimension cross-slots relative to the microparticle diameter, our cross-slot implementation created an extensional flow region that was large enough for agarose hydrogel microparticles to achieve a strain plateau while dwelling near the stagnation point. This strain plateau will be key for accurately and precisely measuring linear viscoelastic properties of small microscale biological objects. The mechanical test was performed in the linear regime, so an analytical mechanical model derived using the elastic-viscoelastic correspondence principle was proposed to extract linear viscoelastic mechanical properties from observed particle strain histories. Particle image velocimetry measurements of the unperturbed velocity field were used to determine where in the device particles experienced extensional flow and the mechanical model should be applied. The measurement throughput in this work was 1 – 2 particles achieving a quasi-steady strain plateau per second, though measurement yield and throughput can be increased with particle-centering upstream device design features. Finally, we provide recommendations for applying the cross-slot microscale creep experiment to other biomaterials and criteria to identify particles that likely achieved a quasi-steady strain state.
{"title":"Microfluidic creep experiment for measuring linear viscoelastic mechanical properties of microparticles in a cross-slot extensional flow device","authors":"Sara Ghanbarpour Mamaghani, Joanna B. Dahl","doi":"10.1101/2024.08.07.607090","DOIUrl":"https://doi.org/10.1101/2024.08.07.607090","url":null,"abstract":"The micromechanical measurement field has struggled to establish repeatable techniques, likely because the deforming stresses can be complicated and difficult to model. Here we demonstrate experimentally the ability of cross-slot microfluidic device to create a quasi-steady deformation state in agarose hydrogel microparticles to replicate a traditional uniaxial creep test at the microscale and at relatively high throughput. A recent numerical study by Lu et al. [Lu, Guo, Yu, Sui. J. Fluid Mech., 2023, 962, A26] showed that viscoelastic capsules flowing through a cross-slot can achieve a quasi-steady strain near the extensional flow stagnation point that is equal to the equilibrium static strain, thereby implying that continuous operation of a cross-slot can accurately capture capsule elastic mechanical behavior in addition to transient behavior. However, no microfluidic cross-slot studies have reported quasi-steady strains for suspended cells or particles, to our knowledge. By using large dimension cross-slots relative to the microparticle diameter, our cross-slot implementation created an extensional flow region that was large enough for agarose hydrogel microparticles to achieve a strain plateau while dwelling near the stagnation point. This strain plateau will be key for accurately and precisely measuring linear viscoelastic properties of small microscale biological objects. The mechanical test was performed in the linear regime, so an analytical mechanical model derived using the elastic-viscoelastic correspondence principle was proposed to extract linear viscoelastic mechanical properties from observed particle strain histories. Particle image velocimetry measurements of the unperturbed velocity field were used to determine where in the device particles experienced extensional flow and the mechanical model should be applied. The measurement throughput in this work was 1 – 2 particles achieving a quasi-steady strain plateau per second, though measurement yield and throughput can be increased with particle-centering upstream device design features. Finally, we provide recommendations for applying the cross-slot microscale creep experiment to other biomaterials and criteria to identify particles that likely achieved a quasi-steady strain state.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141922881","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The growing accessibility of sequencing experiments has significantly accelerated the development of personalized immunotherapies based on the identification of cancer neoantigens. Still, the prediction of neoantigens involves lengthy and inefficient protocols, requiring simultaneous analysis of sequencing data from paired tumor/normal exomes and tumor transcriptome, often resulting in a low success rate. To date, the feasibility of adopting a more efficient strategy has not been fully evaluated. To this end, we developed ENEO, a computational approach to detect cancer neoantigens using solely the tumor RNA-seq data while addressing the lack of matched control through a Bayesian probabilistic model. ENEO was assessed on TESLA benchmark dataset, reporting efficient identification of DNA-alterations derived neoantigens and compelling results against state-of-art exome-based methods. We further validated the method on two independent cohorts, encompassing different tumor types and experimental procedures. Our work demonstrates that a tumor-only RNA-based approach, such as the one implemented in ENEO, maintains accuracy in identifying mutated peptides resulting from expressed genomic alterations, while also broadening the pool of potential pMHCs with RNAspecific mutations in a faster and cost-effective way. ENEO is freely available at https://github.com/ctglab/ENEO
测序实验的普及大大加快了基于癌症新抗原鉴定的个性化免疫疗法的发展。然而，新抗原的预测涉及冗长而低效的方案，需要同时分析配对的肿瘤/正常外显子组和肿瘤转录组的测序数据，往往导致成功率较低。迄今为止，采用更高效策略的可行性尚未得到充分评估。为此，我们开发了ENEO，这是一种仅使用肿瘤RNA-seq数据检测癌症新抗原的计算方法，同时通过贝叶斯概率模型解决了缺乏匹配对照的问题。我们在 TESLA 基准数据集上对 ENEO 进行了评估，结果表明它能有效识别 DNA 改变衍生的新抗原，与基于外显子的先进方法相比，ENEO 的结果令人信服。我们还在两个独立的队列中进一步验证了该方法，这两个队列包括不同的肿瘤类型和实验过程。我们的工作表明，基于肿瘤 RNA 的方法（如 ENEO 中实现的方法）能保持识别基因组表达改变导致的突变肽的准确性，同时还能以更快、更具成本效益的方式扩大具有 RNA 特异性突变的潜在 pMHCs 库。ENEO 可在 https://github.com/ctglab/ENEO 免费获取。
{"title":"Efficient and effective identification of cancer neoantigens from tumor only RNA-seq","authors":"Danilo Tatoni, Mattia Dalsass, Giulia Brunelli, Guido Grandi, Mario Chiariello, Romina D’Aurizio","doi":"10.1101/2024.08.08.607127","DOIUrl":"https://doi.org/10.1101/2024.08.08.607127","url":null,"abstract":"The growing accessibility of sequencing experiments has significantly accelerated the development of personalized immunotherapies based on the identification of cancer neoantigens. Still, the prediction of neoantigens involves lengthy and inefficient protocols, requiring simultaneous analysis of sequencing data from paired tumor/normal exomes and tumor transcriptome, often resulting in a low success rate. To date, the feasibility of adopting a more efficient strategy has not been fully evaluated. To this end, we developed ENEO, a computational approach to detect cancer neoantigens using solely the tumor RNA-seq data while addressing the lack of matched control through a Bayesian probabilistic model. ENEO was assessed on TESLA benchmark dataset, reporting efficient identification of DNA-alterations derived neoantigens and compelling results against state-of-art exome-based methods. We further validated the method on two independent cohorts, encompassing different tumor types and experimental procedures. Our work demonstrates that a tumor-only RNA-based approach, such as the one implemented in ENEO, maintains accuracy in identifying mutated peptides resulting from expressed genomic alterations, while also broadening the pool of potential pMHCs with RNAspecific mutations in a faster and cost-effective way. ENEO is freely available at https://github.com/ctglab/ENEO","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141925182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.607072
O. Maxian, Katrina M Longhini, M. Glotzer
The anterior-posterior axis of Caenorhabditis elegans embryos is determined by the position of sperm entry. The sperm-provided centrosome induces local inhibition of cortical contractility, leading to large-scale myosin flows. This process is driven by the guanine nucleotide exchange factor (GEF) ECT-2, which activates myosin through the GTPase RHO-1. Previously, we showed that in both cell polarization and cytokinesis, Aurora A (AIR-1) is activated on the centrosomes and diffuses to the cortex, where it locally inhibits ECT-2, leading to gradients in myosin concentration. These gradients in turn drive long-range flows that amplify ECT-2 asymmetries (Longhini and Glotzer, 2022). Here, we construct a mathematical model to test whether a minimal set of well characterized, essential elements are necessary and sufficient to explain the spatiotemporal dynamics of AIR-1, ECT-2, and myosin during polarization of the C. elegans model organism. We show that robust establishment of polarity can be obtained in response to a weak AIR-1 signal, and demonstrate the relevance of rapid ECT-2 exchange and a persistent AIR-1 cue during polarization. The tuned model also correctly predicts previously-observed ultrasensitive ECT-2 dynamics during cytokinesis, suggesting that the same minimal circuit operates in both processes.
{"title":"A minimal mathematical model for polarity establishment and centralsplindlin-independent cytokinesis","authors":"O. Maxian, Katrina M Longhini, M. Glotzer","doi":"10.1101/2024.08.07.607072","DOIUrl":"https://doi.org/10.1101/2024.08.07.607072","url":null,"abstract":"The anterior-posterior axis of Caenorhabditis elegans embryos is determined by the position of sperm entry. The sperm-provided centrosome induces local inhibition of cortical contractility, leading to large-scale myosin flows. This process is driven by the guanine nucleotide exchange factor (GEF) ECT-2, which activates myosin through the GTPase RHO-1. Previously, we showed that in both cell polarization and cytokinesis, Aurora A (AIR-1) is activated on the centrosomes and diffuses to the cortex, where it locally inhibits ECT-2, leading to gradients in myosin concentration. These gradients in turn drive long-range flows that amplify ECT-2 asymmetries (Longhini and Glotzer, 2022). Here, we construct a mathematical model to test whether a minimal set of well characterized, essential elements are necessary and sufficient to explain the spatiotemporal dynamics of AIR-1, ECT-2, and myosin during polarization of the C. elegans model organism. We show that robust establishment of polarity can be obtained in response to a weak AIR-1 signal, and demonstrate the relevance of rapid ECT-2 exchange and a persistent AIR-1 cue during polarization. The tuned model also correctly predicts previously-observed ultrasensitive ECT-2 dynamics during cytokinesis, suggesting that the same minimal circuit operates in both processes.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141921613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607151
Annie E Cathignol, L. Kusch, Marianna Angiolelli, E. Troisi Lopez, A. Polverino, A. Romano, G. Sorrentino, V. Jirsa, G. Rabuffo, P. Sorrentino
Complex spontaneous brain dynamics mirror the large number of interactions taking place among regions, supporting higher functions. Such complexity is manifested in the inter-regional dependencies among signals derived from different brain areas, as observed utilising neuroimaging techniques, like magnetoencephalography. The dynamics of this data produce numerous subsets of active regions at any moment as they evolve. Notably, converging evidence shows that these states can be understood in terms of transient coordinated events that spread across the brain over multiple spatial and temporal scales. Those can be used as a proxy of the “effectiveness” of the dynamics, as they become stereotyped or disorganised in neurological diseases. However, given the high dimensional nature of the data, representing them has been challenging thus far. Dimensionality reduction techniques are typically deployed to describe complex interdependencies and improve their interpretability. However, many dimensionality reduction techniques lose information about the sequence of configurations that took place. Here, we leverage a newly described algorithm, PHATE (Potential of Heat-diffusion for Affinity-based Transition Embedding), specifically designed to preserve the dynamics of the system in the low-dimensional embedding space. We analysed source-reconstructed resting-state magnetoencephalography from 18 healthy subjects to represent the dynamics of the configuration in low-dimensional space. After reduction with PHATE, unsupervised clustering via K-means is applied to identify distinct clusters. The topography of the states is described, and the dynamics are represented as a transition matrix. All the results have been checked against null models, providing a parsimonious account of the large-scale, fast, aperiodic dynamics during resting-state.
{"title":"Magnetoencephalography dimensionality reduction informed by dynamic brain states","authors":"Annie E Cathignol, L. Kusch, Marianna Angiolelli, E. Troisi Lopez, A. Polverino, A. Romano, G. Sorrentino, V. Jirsa, G. Rabuffo, P. Sorrentino","doi":"10.1101/2024.08.08.607151","DOIUrl":"https://doi.org/10.1101/2024.08.08.607151","url":null,"abstract":"Complex spontaneous brain dynamics mirror the large number of interactions taking place among regions, supporting higher functions. Such complexity is manifested in the inter-regional dependencies among signals derived from different brain areas, as observed utilising neuroimaging techniques, like magnetoencephalography. The dynamics of this data produce numerous subsets of active regions at any moment as they evolve. Notably, converging evidence shows that these states can be understood in terms of transient coordinated events that spread across the brain over multiple spatial and temporal scales. Those can be used as a proxy of the “effectiveness” of the dynamics, as they become stereotyped or disorganised in neurological diseases. However, given the high dimensional nature of the data, representing them has been challenging thus far. Dimensionality reduction techniques are typically deployed to describe complex interdependencies and improve their interpretability. However, many dimensionality reduction techniques lose information about the sequence of configurations that took place. Here, we leverage a newly described algorithm, PHATE (Potential of Heat-diffusion for Affinity-based Transition Embedding), specifically designed to preserve the dynamics of the system in the low-dimensional embedding space. We analysed source-reconstructed resting-state magnetoencephalography from 18 healthy subjects to represent the dynamics of the configuration in low-dimensional space. After reduction with PHATE, unsupervised clustering via K-means is applied to identify distinct clusters. The topography of the states is described, and the dynamics are represented as a transition matrix. All the results have been checked against null models, providing a parsimonious account of the large-scale, fast, aperiodic dynamics during resting-state.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141922365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.606660
Z. Izadifar, Berenice Charrez, Micaela Almeida, Stijn Robben, K. Pilobello, Janet van der Graaf-Mas, Max Benz, Susan Marquez, Thomas C. Ferrante, K. Shcherbina, Russell Gould, Nina LoGrande, A. Sesay, Donald E Ingber
Despite remarkable advances in Organ-on-a-chip (Organ Chip) microfluidic culture technology, recreating tissue-relevant physiological conditions, such as the region-specific oxygen concentrations, remains a formidable technical challenge, and analysis of tissue functions is commonly carried out using one analytical technique at a time. Here, we describe two-channel Organ Chip microfluidic devices fabricated from polydimethylsiloxane and gas impermeable polycarbonate materials that are integrated with multiple sensors, mounted on a printed circuit board and operated using a commercially available Organ Chip culture instrument. The novelty of this system is that it enables the recreation of physiologically relevant tissue-tissue interfaces and oxygen tension as well as non-invasive continuous measurement of transepithelial electrical resistance, oxygen concentration and pH, combined with simultaneous analysis of cellular metabolic activity (ATP/ADP ratio), cell morphology, and tissue phenotype. We demonstrate the reliable and reproducible functionality of this system in living human Gut and Liver Chip cultures. Changes in tissue barrier function and oxygen tension along with their functional and metabolic responses to chemical stimuli (e.g., calcium chelation, oligomycin) were continuously and noninvasively monitored on-chip for up to 23 days. A physiologically relevant microaerobic microenvironment that supports co-culture of human intestinal cells with living Lactococcus lactis bacteria also was demonstrated in the Gut Chip. The integration of multi-functional sensors into Organ Chips provides a robust and scalable platform for the simultaneous, continuous, and non-invasive monitoring of multiple physiological functions that can significantly enhance the comprehensive and reliable evaluation of engineered tissues in Organ Chip models in basic research, preclinical modeling, and drug development.
{"title":"Organ Chips with integrated multifunctional sensors enable continuous metabolic monitoring at controlled oxygen levels","authors":"Z. Izadifar, Berenice Charrez, Micaela Almeida, Stijn Robben, K. Pilobello, Janet van der Graaf-Mas, Max Benz, Susan Marquez, Thomas C. Ferrante, K. Shcherbina, Russell Gould, Nina LoGrande, A. Sesay, Donald E Ingber","doi":"10.1101/2024.08.08.606660","DOIUrl":"https://doi.org/10.1101/2024.08.08.606660","url":null,"abstract":"Despite remarkable advances in Organ-on-a-chip (Organ Chip) microfluidic culture technology, recreating tissue-relevant physiological conditions, such as the region-specific oxygen concentrations, remains a formidable technical challenge, and analysis of tissue functions is commonly carried out using one analytical technique at a time. Here, we describe two-channel Organ Chip microfluidic devices fabricated from polydimethylsiloxane and gas impermeable polycarbonate materials that are integrated with multiple sensors, mounted on a printed circuit board and operated using a commercially available Organ Chip culture instrument. The novelty of this system is that it enables the recreation of physiologically relevant tissue-tissue interfaces and oxygen tension as well as non-invasive continuous measurement of transepithelial electrical resistance, oxygen concentration and pH, combined with simultaneous analysis of cellular metabolic activity (ATP/ADP ratio), cell morphology, and tissue phenotype. We demonstrate the reliable and reproducible functionality of this system in living human Gut and Liver Chip cultures. Changes in tissue barrier function and oxygen tension along with their functional and metabolic responses to chemical stimuli (e.g., calcium chelation, oligomycin) were continuously and noninvasively monitored on-chip for up to 23 days. A physiologically relevant microaerobic microenvironment that supports co-culture of human intestinal cells with living Lactococcus lactis bacteria also was demonstrated in the Gut Chip. The integration of multi-functional sensors into Organ Chips provides a robust and scalable platform for the simultaneous, continuous, and non-invasive monitoring of multiple physiological functions that can significantly enhance the comprehensive and reliable evaluation of engineered tissues in Organ Chip models in basic research, preclinical modeling, and drug development.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141921932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.607083
Elliott P. Brooks, McKenna R. Casey, Kristen L. Wells, Tsung-Yun Liu, Madeline Van Orman, Lori Sussel
Transcription factors (TFs) are indispensable for maintaining cell identity through regulating cell-specific gene expression. Distinct cell identities derived from a common progenitor are frequently perpetuated by shared TFs; yet the mechanisms that enable these TFs to regulate cell-specific targets are poorly characterized. We report that the TF NKX2.2 is critical for the identity of pancreatic islet α cells by directly activating α cell genes and repressing alternate islet cell fate genes. When compared to the known role of NKX2.2 in islet β cells, we demonstrate that NKX2.2 regulates α cell genes, facilitated in part by α cell specific DNA binding at gene promoters. Furthermore, we have identified the reprogramming factor KLF4 as having enriched expression in α cells, where it co-occupies NKX2.2-bound α cell promoters, is necessary for NKX2.2 promoter occupancy in α cells and co-regulates many NKX2.2 α cell transcriptional targets. Misexpression of Klf4 in β cells is sufficient to manipulate chromatin accessibility, increase binding of NKX2.2 at α cell specific promoter sites, and alter expression of NKX2.2-regulated cell-specific targets. This study identifies KLF4 is a novel α cell factor that cooperates with NKX2.2 to regulate α cell identity.
{"title":"NKX2.2 and KLF4 cooperate to regulate α cell identity","authors":"Elliott P. Brooks, McKenna R. Casey, Kristen L. Wells, Tsung-Yun Liu, Madeline Van Orman, Lori Sussel","doi":"10.1101/2024.08.07.607083","DOIUrl":"https://doi.org/10.1101/2024.08.07.607083","url":null,"abstract":"Transcription factors (TFs) are indispensable for maintaining cell identity through regulating cell-specific gene expression. Distinct cell identities derived from a common progenitor are frequently perpetuated by shared TFs; yet the mechanisms that enable these TFs to regulate cell-specific targets are poorly characterized. We report that the TF NKX2.2 is critical for the identity of pancreatic islet α cells by directly activating α cell genes and repressing alternate islet cell fate genes. When compared to the known role of NKX2.2 in islet β cells, we demonstrate that NKX2.2 regulates α cell genes, facilitated in part by α cell specific DNA binding at gene promoters. Furthermore, we have identified the reprogramming factor KLF4 as having enriched expression in α cells, where it co-occupies NKX2.2-bound α cell promoters, is necessary for NKX2.2 promoter occupancy in α cells and co-regulates many NKX2.2 α cell transcriptional targets. Misexpression of Klf4 in β cells is sufficient to manipulate chromatin accessibility, increase binding of NKX2.2 at α cell specific promoter sites, and alter expression of NKX2.2-regulated cell-specific targets. This study identifies KLF4 is a novel α cell factor that cooperates with NKX2.2 to regulate α cell identity.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141923008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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