Pub Date : 2024-08-08DOI: 10.1101/2024.08.06.606806
Rejin Salimraj, Alessio Perotti, M. Wojewodzic, Dagmar Frisch
The comet assay (CA) was originally developed as toxicity test and quantifies DNA integrity from the distribution of DNA across an electric field. Compromised DNA moves across electric fields faster than intact DNA strands, leaving a quantifiable signature that resembles a comet tail. The dimensions of this comet tail reflect relative DNA damage. We optimized the CA protocol for individual dormant propagules (Single-egg Comet Assay or SE-CA) to inform downstream analyses such as DNA sequencing, of the DNA quality contained in natural genetic archives of past populations. As a model we used dormant eggs of the microcrustacean Daphnia. We tested the SE-CA protocol on impact of processing and storage conditions for dormant eggs and used it to assess DNA damage related to aging of eggs retrieved from recently deposited to centuries-old lake sediment. The SE-CA successfully determined the degree of DNA damage in individual eggs frozen in liquid nitrogen, or at -80°C as well as damage caused by bleaching and historical egg age. In conclusion, our protocol provides a cost-effective method of assessing DNA damage in sedimentary propagules such as dormant Daphnia eggs. More generally, the SE-CA can be applied to test DNA integrity in individual propagules prior to genome sequencing or to quantify environmental impacts on natural sedimentary biobanks.
彗星试验（CA）最初是作为毒性测试而开发的，通过 DNA 在电场中的分布来量化 DNA 的完整性。受损的 DNA 在电场中的移动速度快于完整的 DNA 链，会留下类似彗尾的可量化特征。彗尾的尺寸反映了 DNA 的相对损伤程度。我们优化了针对单个休眠繁殖体的彗星分析方案（单卵彗星分析或SE-CA），以便为DNA测序等下游分析提供信息，了解过去种群天然遗传档案中的DNA质量。我们以微型甲壳动物水蚤的休眠卵为模型。我们测试了 SE-CA 方案对休眠卵的处理和储存条件的影响，并用它来评估从新近沉积到数百年历史的湖泊沉积物中提取的卵的与老化有关的 DNA 损伤。SE-CA 成功地确定了在液氮或-80°C条件下冷冻的单个卵的 DNA 损伤程度，以及漂白和历史卵龄造成的损伤。总之，我们的方案为评估休眠水蚤卵等沉积繁殖体的 DNA 损伤提供了一种经济有效的方法。更广泛地说，SE-CA 可用于在基因组测序前检测单个繁殖体的 DNA 完整性，或量化环境对天然沉积生物库的影响。
{"title":"Single-egg Comet Assay: a protocol to quantify DNA damage in aquatic dormant stages","authors":"Rejin Salimraj, Alessio Perotti, M. Wojewodzic, Dagmar Frisch","doi":"10.1101/2024.08.06.606806","DOIUrl":"https://doi.org/10.1101/2024.08.06.606806","url":null,"abstract":"The comet assay (CA) was originally developed as toxicity test and quantifies DNA integrity from the distribution of DNA across an electric field. Compromised DNA moves across electric fields faster than intact DNA strands, leaving a quantifiable signature that resembles a comet tail. The dimensions of this comet tail reflect relative DNA damage. We optimized the CA protocol for individual dormant propagules (Single-egg Comet Assay or SE-CA) to inform downstream analyses such as DNA sequencing, of the DNA quality contained in natural genetic archives of past populations. As a model we used dormant eggs of the microcrustacean Daphnia. We tested the SE-CA protocol on impact of processing and storage conditions for dormant eggs and used it to assess DNA damage related to aging of eggs retrieved from recently deposited to centuries-old lake sediment. The SE-CA successfully determined the degree of DNA damage in individual eggs frozen in liquid nitrogen, or at -80°C as well as damage caused by bleaching and historical egg age. In conclusion, our protocol provides a cost-effective method of assessing DNA damage in sedimentary propagules such as dormant Daphnia eggs. More generally, the SE-CA can be applied to test DNA integrity in individual propagules prior to genome sequencing or to quantify environmental impacts on natural sedimentary biobanks.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141926548","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 : 2024-08-08DOI: 10.1101/2024.08.06.606744
Syed Mashab Ali Shah, Fazal Haq, Kunxuan Huang, Qi Wang, Linlin Liu, Ying Li, Yong Wang, Asaf Khan, Ruihuan Yang, M. Khojasteh, Xiameng Xu, Zhengyin Xu, Gongyou Chen
Bacterial Blight of Cotton (BBC) caused by Xanthomonas citri pv. malvacearum (Xcm) is an important and destructive disease affecting cotton plants. Transcription activator-like effectors (TALEs) released by the pathogen regulate cotton resistance to the susceptibility. In this study, we sequenced the whole genome of Xcm Xss-V2-18 and identified eight tal genes; seven on the plasmids and one on the chromosome. Deletion and complementation experiments of Xss-V2-18 tal genes demonstrated that Tal1b is required for full virulence on cotton. Transcriptome profiling coupled with TALE-binding element prediction revealed that Tal1b targets GhSWEET15A04/D04 and GhSWEET15D02 simultaneously. Expression analysis confirmed the independent inducibility of GhSWEET15A04/D04 and GhSWEET15D02 by Tal1b, whereas GhSWEET15A04/D04 is additionally targeted by Tal1. Moreover, GUS (β-glucuronidase) and Xa10-mediated HR (hypersensitive response) assays indicated that the EBEs are required for the direct and specific activation of the candidate targets by Tal1 and Ta1b. These findings may advance our understanding of the dynamics between TALEs and EBEs, and decipher a simple and effective DNA-binding mechanism that could lead to the development of more efficient methods for gene editing and transgenic research.
由柠檬黄单胞菌（Xanthomonas citri pv. malvacearum，Xcm）引起的棉花细菌性疫病（Bacterial Blight of Cotton，BBC）是影响棉花植株的一种重要的破坏性病害。病原体释放的转录激活剂样效应子（TALEs）可调节棉花的抗病性和易感性。在这项研究中，我们对 Xcm Xss-V2-18 的全基因组进行了测序，发现了 8 个 tal 基因，其中 7 个在质粒上，1 个在染色体上。Xss-V2-18 tal 基因的缺失和互补实验表明，Tal1b 是棉花上完全毒力所必需的。转录组分析和 TALE 结合元件预测显示，Tal1b 同时以 GhSWEET15A04/D04 和 GhSWEET15D02 为靶标。表达分析证实了 Tal1b 对 GhSWEET15A04/D04 和 GhSWEET15D02 的独立诱导性，而 GhSWEET15A04/D04 则是 Tal1 的额外靶标。此外，GUS（β-葡糖醛酸酶）和 Xa10 介导的 HR（超敏反应）测定表明，Tal1 和 Ta1b 直接特异性激活候选靶标需要 EBE。这些发现可能会促进我们对 TALEs 和 EBEs 之间动态的了解，并破译一种简单有效的 DNA 结合机制，从而开发出更有效的基因编辑和转基因研究方法。
{"title":"Two TAL effectors of Xanthomonas citri pv. malvacearum target GhSWEET15 as the susceptibility genes for bacterial blight of cotton","authors":"Syed Mashab Ali Shah, Fazal Haq, Kunxuan Huang, Qi Wang, Linlin Liu, Ying Li, Yong Wang, Asaf Khan, Ruihuan Yang, M. Khojasteh, Xiameng Xu, Zhengyin Xu, Gongyou Chen","doi":"10.1101/2024.08.06.606744","DOIUrl":"https://doi.org/10.1101/2024.08.06.606744","url":null,"abstract":"Bacterial Blight of Cotton (BBC) caused by Xanthomonas citri pv. malvacearum (Xcm) is an important and destructive disease affecting cotton plants. Transcription activator-like effectors (TALEs) released by the pathogen regulate cotton resistance to the susceptibility. In this study, we sequenced the whole genome of Xcm Xss-V2-18 and identified eight tal genes; seven on the plasmids and one on the chromosome. Deletion and complementation experiments of Xss-V2-18 tal genes demonstrated that Tal1b is required for full virulence on cotton. Transcriptome profiling coupled with TALE-binding element prediction revealed that Tal1b targets GhSWEET15A04/D04 and GhSWEET15D02 simultaneously. Expression analysis confirmed the independent inducibility of GhSWEET15A04/D04 and GhSWEET15D02 by Tal1b, whereas GhSWEET15A04/D04 is additionally targeted by Tal1. Moreover, GUS (β-glucuronidase) and Xa10-mediated HR (hypersensitive response) assays indicated that the EBEs are required for the direct and specific activation of the candidate targets by Tal1 and Ta1b. These findings may advance our understanding of the dynamics between TALEs and EBEs, and decipher a simple and effective DNA-binding mechanism that could lead to the development of more efficient methods for gene editing and transgenic research.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141925623","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 : 2024-08-08DOI: 10.1101/2024.08.06.606834
Hanna Liao, Sanjay Kottapalli, Yuqi Huang, Matthew Chaw, Jase Gehring, Olivia Waltner, Melissa Phung-Rojas, R. Daza, Frederick A. Matsen, C. Trapnell, Jay Shendure, Sanjay R Srivatsan
Spatial genomic technologies include imaging- and sequencing-based methods (1–3). An emerging subcategory of sequencing-based methods relies on a surface coated with coordinate-associated DNA barcodes, which are leveraged to tag endogenous nucleic acids or cells in an overlaid tissue section (4–7). However, the physical registration of DNA barcodes to spatial coordinates is challenging, necessitating either high density printing of coordinate-specific oligonucleotides or in situ sequencing/probing of randomly deposited, oligonucleotide-bearing beads. As a consequence, the surface areas available to sequencing-based spatial genomic methods are constrained by the time, labor, cost, and instrumentation required to either print, synthesize or decode a coordinate-tagged surface. To address this challenge, we developed SCOPE (Spatial reConstruction via Oligonucleotide Proximity Encoding), an optics-free, DNA microscopy (8) inspired method. With SCOPE, the relative positions of randomly deposited beads on a 2D surface are inferred from the ex situ sequencing of chimeric molecules formed from diffusing “sender” and tethered “receiver” oligonucleotides. As a first proof-of-concept, we apply SCOPE to reconstruct an asymmetric “swoosh” shape resembling the Nike logo (16.75 × 9.25 mm). Next, we use a microarray printer to encode a “color” version of the Snellen eye chart for visual acuity (17.18 × 40.97 mm), and apply SCOPE to achieve optics-free reconstruction of individual letters. Although these are early demonstrations of the concept and much work remains to be done, we envision that the optics-free, sequencing-based quantitation of the molecular proximities of DNA barcodes will enable spatial genomics in constant experimental time, across fields of view and at resolutions that are determined by sequencing depth, bead size, and diffusion kinetics, rather than the limitations of optical instruments or microarray printers.
空间基因组技术包括基于成像和测序的方法（1-3）。基于测序方法的一个新兴子类别依赖于涂有坐标相关 DNA 条形码的表面，利用这些条形码标记覆盖组织切片中的内源性核酸或细胞（4-7）。然而，DNA 条形码与空间坐标的物理配准具有挑战性，需要高密度打印坐标特异性寡核苷酸，或对随机沉积的含有寡核苷酸的珠子进行原位测序/探针。因此，基于测序的空间基因组学方法可用的表面区域受到打印、合成或解码坐标标记表面所需的时间、人力、成本和仪器的限制。为了应对这一挑战，我们开发了 SCOPE（通过寡核苷酸邻近编码进行空间重构），这是一种受 DNA 显微镜（8）启发的无光学方法。利用 SCOPE，可以通过对扩散的 "发送者 "和系留的 "接收者 "寡核苷酸形成的嵌合分子进行原位测序，推断出二维表面上随机沉积的珠子的相对位置。作为第一个概念验证，我们应用 SCOPE 重建了一个类似耐克标志（16.75 × 9.25 毫米）的不对称 "咻 "形。接下来，我们使用微阵列打印机对 "彩色 "版的斯奈伦视力表（17.18 × 40.97 毫米）进行编码，并应用 SCOPE 实现单个字母的无光学重建。尽管这些只是对这一概念的早期演示，还有许多工作要做，但我们设想，基于无光学的测序对 DNA 条形码的分子邻近性进行定量，将能在恒定的实验时间内实现跨视场的空间基因组学，其分辨率由测序深度、珠子大小和扩散动力学决定，而不是受光学仪器或微阵列打印机的限制。
{"title":"Optics-free reconstruction of 2D images via DNA barcode proximity graphs","authors":"Hanna Liao, Sanjay Kottapalli, Yuqi Huang, Matthew Chaw, Jase Gehring, Olivia Waltner, Melissa Phung-Rojas, R. Daza, Frederick A. Matsen, C. Trapnell, Jay Shendure, Sanjay R Srivatsan","doi":"10.1101/2024.08.06.606834","DOIUrl":"https://doi.org/10.1101/2024.08.06.606834","url":null,"abstract":"Spatial genomic technologies include imaging- and sequencing-based methods (1–3). An emerging subcategory of sequencing-based methods relies on a surface coated with coordinate-associated DNA barcodes, which are leveraged to tag endogenous nucleic acids or cells in an overlaid tissue section (4–7). However, the physical registration of DNA barcodes to spatial coordinates is challenging, necessitating either high density printing of coordinate-specific oligonucleotides or in situ sequencing/probing of randomly deposited, oligonucleotide-bearing beads. As a consequence, the surface areas available to sequencing-based spatial genomic methods are constrained by the time, labor, cost, and instrumentation required to either print, synthesize or decode a coordinate-tagged surface. To address this challenge, we developed SCOPE (Spatial reConstruction via Oligonucleotide Proximity Encoding), an optics-free, DNA microscopy (8) inspired method. With SCOPE, the relative positions of randomly deposited beads on a 2D surface are inferred from the ex situ sequencing of chimeric molecules formed from diffusing “sender” and tethered “receiver” oligonucleotides. As a first proof-of-concept, we apply SCOPE to reconstruct an asymmetric “swoosh” shape resembling the Nike logo (16.75 × 9.25 mm). Next, we use a microarray printer to encode a “color” version of the Snellen eye chart for visual acuity (17.18 × 40.97 mm), and apply SCOPE to achieve optics-free reconstruction of individual letters. Although these are early demonstrations of the concept and much work remains to be done, we envision that the optics-free, sequencing-based quantitation of the molecular proximities of DNA barcodes will enable spatial genomics in constant experimental time, across fields of view and at resolutions that are determined by sequencing depth, bead size, and diffusion kinetics, rather than the limitations of optical instruments or microarray printers.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141926950","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 : 2024-08-08DOI: 10.1101/2024.08.06.606645
Adrian Aleman-Zapata, Melisa Maidana Capitan, Anumita Samanta, Pelin Özsezer, K. Agarwal, Tugdual Adam, Abdelrahman Rayan, Lisa Genzel
Cannabidiol (CBD), increasingly recognized for its potential to treat insomnia, notably extends NonREM sleep phases and modifies sleep-associated ripple dynamics. Utilizing a threshold-based approach, our study differentiated distinct ripple types in rats, clarifying the contributions of intra-hippocampal (CA3) and cortical (mEC) regions to these events. The findings reveal that CBD primarily influences the CA3’s input to the CA1, resulting in an increased occurrence of short ripples predominantly induced by cortical (mEC) activity and a corresponding decrease in long, intra-hippocampal sharp-wave-ripples. This study highlights the critical interplay between the CA3 and entorhinal cortex dynamics in shaping the characteristics of hippocampal ripples under the influence of CBD.
{"title":"Differential Contributions of CA3 and Entorhinal Cortex Inputs to Ripple Patterns in the Hippocampus Under Cannabidiol","authors":"Adrian Aleman-Zapata, Melisa Maidana Capitan, Anumita Samanta, Pelin Özsezer, K. Agarwal, Tugdual Adam, Abdelrahman Rayan, Lisa Genzel","doi":"10.1101/2024.08.06.606645","DOIUrl":"https://doi.org/10.1101/2024.08.06.606645","url":null,"abstract":"Cannabidiol (CBD), increasingly recognized for its potential to treat insomnia, notably extends NonREM sleep phases and modifies sleep-associated ripple dynamics. Utilizing a threshold-based approach, our study differentiated distinct ripple types in rats, clarifying the contributions of intra-hippocampal (CA3) and cortical (mEC) regions to these events. The findings reveal that CBD primarily influences the CA3’s input to the CA1, resulting in an increased occurrence of short ripples predominantly induced by cortical (mEC) activity and a corresponding decrease in long, intra-hippocampal sharp-wave-ripples. This study highlights the critical interplay between the CA3 and entorhinal cortex dynamics in shaping the characteristics of hippocampal ripples under the influence of CBD.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141927715","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 : 2024-08-08DOI: 10.1101/2024.08.06.606930
Rakshanya Sekar, Adrian V. Cioanca, Yilei (Evelyn) Yang, K. S. Kamath, Luke Carroll, Riccardo Natoli, Yvette Wooff
Neuroinflammation is a pathological process mediated through immune cell activation and pro-inflammatory cytokine release, resulting in neuronal cell death. In the central nervous system (CNS), neuroinflammation is a characteristic feature underlying the onset and progression of retinal and neurodegenerative diseases. Targeting neuroinflammation to reduce neuronal cell death and protect against visual and cognitive declines is therefore a key therapeutic strategy. However, due to the complex and multi-faceted nature of these diseases, to date there has been little therapeutic success with single target approaches insufficient to tackle widespread and multi-pathway inflammatory cascades. Furthermore, as the retina and brain reside within immune-privileged environments, a major challenge in treating these diseases is producing and delivering a therapeutic that, in itself, does not exacerbate inflammation. Extracellular vesicles (EV), derived from red blood cells (RBC EV), present a promising solution to overcome these hurdles, due to their innate ability to cross blood-tissue barriers, biocompatible nature, and their broad anti-inflammatory properties to modulate complex neuroinflammatory pathways. This study therefore investigated the therapeutic potential of RBC EV in mediating neuroinflammation using an in-vivo photo-oxidative damage model of retinal degeneration as a model for CNS neuroinflammation. In this work, we developed a novel incubation pipeline using N1 medium supplement and superoxide dismutase (SOD) supplementation to promote the production of safe, neuroprotective, and anti-inflammatory RBC EV. Delivery of RBC EV in vivo, was shown to be safe with strong penetration across all retinal layers. Further, therapeutic administration of RBC EV via local intravitreal injection significantly reduced inflammation and cell death and preserved retinal function. Notably, strong safety and therapeutic efficacy was also demonstrated in the retina following systemic (intraperitoneal) administration, highlighting a potential game-changing approach for less-invasive therapeutic delivery to the CNS. Finally, multi-omic analyses and in vitro findings supported an anti-inflammatory mechanism-of-action, with RBC EV modulating pro-inflammatory cytokine release, including those known to be involved in the pathogenesis of retinal and neurodegenerative diseases. Taken together, these findings highlight the broad applicability of RBC EV in treating neuroinflammation in the CNS, presenting a scalable and effective treatment approach for these currently untreatable diseases.
神经炎症是通过免疫细胞激活和促炎细胞因子释放介导的病理过程，导致神经细胞死亡。在中枢神经系统（CNS）中，神经炎症是视网膜和神经退行性疾病发病和发展的一个特征。因此，以神经炎症为靶点，减少神经元细胞死亡，防止视觉和认知能力下降，是一项关键的治疗策略。然而，由于这些疾病的复杂性和多面性，迄今为止，单一靶点方法不足以解决广泛和多途径的炎症级联，治疗效果甚微。此外，由于视网膜和大脑处于免疫优势环境中，治疗这些疾病的一个主要挑战是生产和提供一种本身不会加剧炎症的疗法。从红细胞（RBC EV）中提取的细胞外囊泡（EV）是克服这些障碍的一个很有前景的解决方案，因为它们具有天生的穿越血液-组织屏障的能力、生物相容性以及广泛的抗炎特性，可以调节复杂的神经炎症通路。因此，本研究利用视网膜变性的体内光氧化损伤模型作为中枢神经系统神经炎症的模型，研究了 RBC EV 在介导神经炎症方面的治疗潜力。在这项工作中，我们利用 N1 培养基补充剂和超氧化物歧化酶（SOD）补充剂开发了一种新型孵育管道，以促进安全、神经保护性和抗炎性 RBC EV 的产生。研究表明，RBC EV 在体内的输送是安全的，并能在视网膜各层牢固渗透。此外，通过局部玻璃体内注射 RBC EV 治疗用药可显著减少炎症和细胞死亡，保护视网膜功能。值得注意的是，全身（腹膜内）给药后在视网膜上也显示出很强的安全性和疗效，这突显了一种潜在的改变游戏规则的方法，即向中枢神经系统提供微创治疗。最后，多组学分析和体外研究结果支持抗炎作用机制，RBC EV 可调节促炎细胞因子的释放，包括那些已知参与视网膜和神经退行性疾病发病机制的细胞因子。综上所述，这些研究结果凸显了 RBC EV 在治疗中枢神经系统神经炎症方面的广泛适用性，为这些目前无法治疗的疾病提供了一种可扩展的有效治疗方法。
{"title":"Therapeutic potential of red blood cell-derived extracellular vesicles in reducing neuroinflammation and protecting against retinal degeneration","authors":"Rakshanya Sekar, Adrian V. Cioanca, Yilei (Evelyn) Yang, K. S. Kamath, Luke Carroll, Riccardo Natoli, Yvette Wooff","doi":"10.1101/2024.08.06.606930","DOIUrl":"https://doi.org/10.1101/2024.08.06.606930","url":null,"abstract":"Neuroinflammation is a pathological process mediated through immune cell activation and pro-inflammatory cytokine release, resulting in neuronal cell death. In the central nervous system (CNS), neuroinflammation is a characteristic feature underlying the onset and progression of retinal and neurodegenerative diseases. Targeting neuroinflammation to reduce neuronal cell death and protect against visual and cognitive declines is therefore a key therapeutic strategy. However, due to the complex and multi-faceted nature of these diseases, to date there has been little therapeutic success with single target approaches insufficient to tackle widespread and multi-pathway inflammatory cascades. Furthermore, as the retina and brain reside within immune-privileged environments, a major challenge in treating these diseases is producing and delivering a therapeutic that, in itself, does not exacerbate inflammation. Extracellular vesicles (EV), derived from red blood cells (RBC EV), present a promising solution to overcome these hurdles, due to their innate ability to cross blood-tissue barriers, biocompatible nature, and their broad anti-inflammatory properties to modulate complex neuroinflammatory pathways. This study therefore investigated the therapeutic potential of RBC EV in mediating neuroinflammation using an in-vivo photo-oxidative damage model of retinal degeneration as a model for CNS neuroinflammation. In this work, we developed a novel incubation pipeline using N1 medium supplement and superoxide dismutase (SOD) supplementation to promote the production of safe, neuroprotective, and anti-inflammatory RBC EV. Delivery of RBC EV in vivo, was shown to be safe with strong penetration across all retinal layers. Further, therapeutic administration of RBC EV via local intravitreal injection significantly reduced inflammation and cell death and preserved retinal function. Notably, strong safety and therapeutic efficacy was also demonstrated in the retina following systemic (intraperitoneal) administration, highlighting a potential game-changing approach for less-invasive therapeutic delivery to the CNS. Finally, multi-omic analyses and in vitro findings supported an anti-inflammatory mechanism-of-action, with RBC EV modulating pro-inflammatory cytokine release, including those known to be involved in the pathogenesis of retinal and neurodegenerative diseases. Taken together, these findings highlight the broad applicability of RBC EV in treating neuroinflammation in the CNS, presenting a scalable and effective treatment approach for these currently untreatable diseases.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141927733","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 : 2024-08-08DOI: 10.1101/2024.08.07.606952
Luciana de Gennaro, Matteo Burgio, G. Lacalandra, Francesco Petronella, Alberto L’Abbate, Francesco Ravasini, B. Trombetta, Annalisa Rizzo, Mario Ventura, Vincenzo Cicirelli
Background Disorders of Sexual Development (DSD) in dogs, similar to humans, arise from irregularities in genetic determinants, gonadal differentiation, or phenotypic sex development. The French Bulldog, a breed that has seen a surge in popularity and demand, has also shown a marked increase in DSD incidence. This study aims to characterize the genetic underpinnings of DSD in a French Bulldog named Brutus, exhibiting ambiguous genitalia and internal sexual anatomy, and to explore the impact of breeding practices on genetic diversity within the breed. Methods We utilized a comprehensive approach combining conventional cytogenetics, molecular techniques, and deep sequencing to investigate the genetic profile of Brutus. The sequence data were compared to three other male French Bulldogs genome sequences with typical reproductive anatomy, including Brutus’s father, and the canine reference genome (CanFam6). Findings Our findings revealed a 22% mosaicism (78, XX/77, XX), the absence of the SRY gene, and the presence of 43 unique Single Nucleotide Variants (SNVs) not inherited from the father. Notably, the Run of Homozygosity (ROH) analysis showed Brutus has a significantly higher number of homozygous segments compared to other Bulldogs, with a total length of these fragments 50% greater than the average, strongly suggesting this dog is the product of the mating between siblings. While no direct causative genes for the DSD phenotype were identified four candidate loci warranting further investigation were highlighted. Conclusions Our study highlighted the need for a better annotated and curated reference dog genome to define genes causative of any specific phenotype, suggests a potential genetic basis for the DSD phenotype in dogs, and underscores the consequences of uncontrolled breeding practices in French Bulldogs. These findings highlight the importance of implementing strategic genetic management to preserve genetic health and diversity in canine populations.
{"title":"Genomic sequencing to detect cross-breeding quality in dogs: an example studying disorders in sexual development","authors":"Luciana de Gennaro, Matteo Burgio, G. Lacalandra, Francesco Petronella, Alberto L’Abbate, Francesco Ravasini, B. Trombetta, Annalisa Rizzo, Mario Ventura, Vincenzo Cicirelli","doi":"10.1101/2024.08.07.606952","DOIUrl":"https://doi.org/10.1101/2024.08.07.606952","url":null,"abstract":"Background Disorders of Sexual Development (DSD) in dogs, similar to humans, arise from irregularities in genetic determinants, gonadal differentiation, or phenotypic sex development. The French Bulldog, a breed that has seen a surge in popularity and demand, has also shown a marked increase in DSD incidence. This study aims to characterize the genetic underpinnings of DSD in a French Bulldog named Brutus, exhibiting ambiguous genitalia and internal sexual anatomy, and to explore the impact of breeding practices on genetic diversity within the breed. Methods We utilized a comprehensive approach combining conventional cytogenetics, molecular techniques, and deep sequencing to investigate the genetic profile of Brutus. The sequence data were compared to three other male French Bulldogs genome sequences with typical reproductive anatomy, including Brutus’s father, and the canine reference genome (CanFam6). Findings Our findings revealed a 22% mosaicism (78, XX/77, XX), the absence of the SRY gene, and the presence of 43 unique Single Nucleotide Variants (SNVs) not inherited from the father. Notably, the Run of Homozygosity (ROH) analysis showed Brutus has a significantly higher number of homozygous segments compared to other Bulldogs, with a total length of these fragments 50% greater than the average, strongly suggesting this dog is the product of the mating between siblings. While no direct causative genes for the DSD phenotype were identified four candidate loci warranting further investigation were highlighted. Conclusions Our study highlighted the need for a better annotated and curated reference dog genome to define genes causative of any specific phenotype, suggests a potential genetic basis for the DSD phenotype in dogs, and underscores the consequences of uncontrolled breeding practices in French Bulldogs. These findings highlight the importance of implementing strategic genetic management to preserve genetic health and diversity in canine populations.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141929692","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 : 2024-08-08DOI: 10.1101/2024.08.06.606897
David H. Perez, Daniel Antfolk, Elliot Medina, David Gonzalez-Perez, Vincent C. Luca
Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although several Notch inhibitors are under development as cancer therapies, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins that mimic the tension-generating mechanism of endogenous ligands. SNAGs were designed by fusing a high-affinity variant of the Notch ligand Delta-like 4 (DLL4) to antibody fragments that induce target internalization. This bispecific format enables the SNAG-bound biomarkers to “pull” on Notch receptors, triggering Notch activation in mixed populations of biomarker-expressing and non-expressing cells. SNAGs targeting the immune checkpoint PDL1 potently activated Notch in co-cultures of Notch1-and PDL1-expressing cells, but not in monocultures of Notch1-expressing cells alone. Additional SNAGs targeting the tumor antigens CD19 and HER2 also activated Notch in mixed cell populations, indicating that the SNAG design concept is adaptable to multiple biomarkers. SNAG-mediated Notch activation was blocked by a dynamin inhibitor, and efficacy increased dramatically when SNAGs were dimerized via fusion to antibody Fc domains, suggesting that endocytosis and multimerization are important for optimal SNAG function. These insights will greatly expand our ability to modulate Notch signaling for applications in immunotherapy and regenerative medicine.
{"title":"Engineering synthetic agonists for targeted activation of Notch signaling","authors":"David H. Perez, Daniel Antfolk, Elliot Medina, David Gonzalez-Perez, Vincent C. Luca","doi":"10.1101/2024.08.06.606897","DOIUrl":"https://doi.org/10.1101/2024.08.06.606897","url":null,"abstract":"Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although several Notch inhibitors are under development as cancer therapies, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins that mimic the tension-generating mechanism of endogenous ligands. SNAGs were designed by fusing a high-affinity variant of the Notch ligand Delta-like 4 (DLL4) to antibody fragments that induce target internalization. This bispecific format enables the SNAG-bound biomarkers to “pull” on Notch receptors, triggering Notch activation in mixed populations of biomarker-expressing and non-expressing cells. SNAGs targeting the immune checkpoint PDL1 potently activated Notch in co-cultures of Notch1-and PDL1-expressing cells, but not in monocultures of Notch1-expressing cells alone. Additional SNAGs targeting the tumor antigens CD19 and HER2 also activated Notch in mixed cell populations, indicating that the SNAG design concept is adaptable to multiple biomarkers. SNAG-mediated Notch activation was blocked by a dynamin inhibitor, and efficacy increased dramatically when SNAGs were dimerized via fusion to antibody Fc domains, suggesting that endocytosis and multimerization are important for optimal SNAG function. These insights will greatly expand our ability to modulate Notch signaling for applications in immunotherapy and regenerative medicine.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141927535","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 : 2024-08-08DOI: 10.1101/2024.08.07.606969
Tianshu Li, Giancarlo La Camera
Fitting a hidden Markov Model (HMM) to neural data is a powerful method to segment a spatiotemporal stream of neural activity into sequences of discrete hidden states. Application of HMM has allowed to uncover hidden states and signatures of neural dynamics that seem relevant for sensory and cognitive processes. This has been accomplished especially in datasets comprising ensembles of simultaneously recorded cortical spike trains. However, the HMM analysis of spike data is involved and requires a careful handling of model selection. Two main issues are: (i) the cross-validated likelihood function typically increases with the number of hidden states; (ii) decoding the data with an HMM can lead to very rapid state switching due to fast oscillations in state probabilities. The first problem is related to the phenomenon of over-segmentation and leads to overfitting. The second problem is at odds with the empirical fact that hidden states in cortex tend to last from hundred of milliseconds to seconds. Here, we show that we can alleviate both problems by regularizing a Poisson-HMM during training so as to enforce large self-transition probabilities. We call this algorithm the ‘sticky Poisson-HMM’ (sPHMM). When used to-gether with the Bayesian Information Criterion for model selection, the sPHMM successfully eliminates rapid state switching, outperforming an alternative strategy based on an HMM with a large prior on the self-transition probabilities. The sPHMM also captures the ground truth in surrogate datasets built to resemble the statistical properties of the experimental data.
{"title":"A sticky Poisson Hidden Markov Model for spike data","authors":"Tianshu Li, Giancarlo La Camera","doi":"10.1101/2024.08.07.606969","DOIUrl":"https://doi.org/10.1101/2024.08.07.606969","url":null,"abstract":"Fitting a hidden Markov Model (HMM) to neural data is a powerful method to segment a spatiotemporal stream of neural activity into sequences of discrete hidden states. Application of HMM has allowed to uncover hidden states and signatures of neural dynamics that seem relevant for sensory and cognitive processes. This has been accomplished especially in datasets comprising ensembles of simultaneously recorded cortical spike trains. However, the HMM analysis of spike data is involved and requires a careful handling of model selection. Two main issues are: (i) the cross-validated likelihood function typically increases with the number of hidden states; (ii) decoding the data with an HMM can lead to very rapid state switching due to fast oscillations in state probabilities. The first problem is related to the phenomenon of over-segmentation and leads to overfitting. The second problem is at odds with the empirical fact that hidden states in cortex tend to last from hundred of milliseconds to seconds. Here, we show that we can alleviate both problems by regularizing a Poisson-HMM during training so as to enforce large self-transition probabilities. We call this algorithm the ‘sticky Poisson-HMM’ (sPHMM). When used to-gether with the Bayesian Information Criterion for model selection, the sPHMM successfully eliminates rapid state switching, outperforming an alternative strategy based on an HMM with a large prior on the self-transition probabilities. The sPHMM also captures the ground truth in surrogate datasets built to resemble the statistical properties of the experimental data.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141925917","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 : 2024-08-08DOI: 10.1101/2024.08.06.606828
R. N. Platt, Egie E. Enabulele, Ehizogie Adeyemi, Marian O Agbugui, OG Ajakaye, E. C. Amaechi, Chika E Ejikeugwu, Christopher Igbeneghu, V. Njom, Precious Dlamini, Grace-Ann Arya, Robbie Diaz, M. Rabone, F. Allan, Bonnie Webster, A. Emery, David Rollinson, Timothy J.C. Anderson
The human parasitic fluke, Schistosoma haematobium hybridizes with the livestock parasite S. bovis in the laboratory, but the extent of hybridization in nature is unclear. We analyzed 34.6 million single nucleotide variants in 162 samples from 18 African countries, revealing a sharp genetic discontinuity between northern and southern S. haematobium. We found no evidence for recent hybridization. Instead the data reveal admixture events that occurred 257-879 generations ago in northern S. haematobium populations. Fifteen introgressed S. bovis genes are approaching fixation in northern S. haematobium with four genes potentially driving adaptation. We identified 19 regions that were resistant to introgression; these were enriched on the sex chromosomes. These results (i) demonstrate strong barriers to gene flow between these species, (ii) indicate that hybridization may be less common than currently envisaged, but (iii) reveal profound genomic consequences of interspecific hybridization between schistosomes of medical and veterinary importance.
{"title":"Genomic data reveal a north-south split and introgression history of blood fluke (Schistosoma haematobium) populations from across Africa","authors":"R. N. Platt, Egie E. Enabulele, Ehizogie Adeyemi, Marian O Agbugui, OG Ajakaye, E. C. Amaechi, Chika E Ejikeugwu, Christopher Igbeneghu, V. Njom, Precious Dlamini, Grace-Ann Arya, Robbie Diaz, M. Rabone, F. Allan, Bonnie Webster, A. Emery, David Rollinson, Timothy J.C. Anderson","doi":"10.1101/2024.08.06.606828","DOIUrl":"https://doi.org/10.1101/2024.08.06.606828","url":null,"abstract":"The human parasitic fluke, Schistosoma haematobium hybridizes with the livestock parasite S. bovis in the laboratory, but the extent of hybridization in nature is unclear. We analyzed 34.6 million single nucleotide variants in 162 samples from 18 African countries, revealing a sharp genetic discontinuity between northern and southern S. haematobium. We found no evidence for recent hybridization. Instead the data reveal admixture events that occurred 257-879 generations ago in northern S. haematobium populations. Fifteen introgressed S. bovis genes are approaching fixation in northern S. haematobium with four genes potentially driving adaptation. We identified 19 regions that were resistant to introgression; these were enriched on the sex chromosomes. These results (i) demonstrate strong barriers to gene flow between these species, (ii) indicate that hybridization may be less common than currently envisaged, but (iii) reveal profound genomic consequences of interspecific hybridization between schistosomes of medical and veterinary importance.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141927497","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 : 2024-08-08DOI: 10.1101/2024.08.08.606782
D. F. Mega, Parul Sharma, Anja Kipar, U. Hetzel, Chloe Bramwell, Alan Merritt, Samuel Wright, Chris Plummer, R. Urbanowicz, James P. Stewart
Seaweed derived compounds are a renewable resource utilised in the manufacturing and food industry. This study focuses on an Enriched seaweed extract (ESE) isolated from Ascophyllum nodosum. ESE was screened for antiviral activity by plaque reduction assays against influenza A viruses (IAV) H1N1 and H3N2 subtypes. Time of addition assays and FACS analysis were used to help determine the modes of action. The therapeutic potential of ESE was then explored using differentiated human bronchiole epithelial cells at the air liquid interphase and a murine model challenged with IAV. The data indicates ESE primarily interacts directly with virions, preventing virus cell binding. Interestingly, ESE also inhibits early and late stage of the influenza A lifecycle when treatment occurs after cell binding. This inhibitory effect appears to prevent internalisation of virus and release of progeny virus by targeting neuraminidase activity. Intranasal administration of ESE in mice infected with IAV reduced viral load in lung tissue. ESE may be a promising broad acting antiviral agent in the treatment of influenza infections.
{"title":"Phlorotannin rich Ascophyllum nodosum seaweed extract inhibits influenza infection","authors":"D. F. Mega, Parul Sharma, Anja Kipar, U. Hetzel, Chloe Bramwell, Alan Merritt, Samuel Wright, Chris Plummer, R. Urbanowicz, James P. Stewart","doi":"10.1101/2024.08.08.606782","DOIUrl":"https://doi.org/10.1101/2024.08.08.606782","url":null,"abstract":"Seaweed derived compounds are a renewable resource utilised in the manufacturing and food industry. This study focuses on an Enriched seaweed extract (ESE) isolated from Ascophyllum nodosum. ESE was screened for antiviral activity by plaque reduction assays against influenza A viruses (IAV) H1N1 and H3N2 subtypes. Time of addition assays and FACS analysis were used to help determine the modes of action. The therapeutic potential of ESE was then explored using differentiated human bronchiole epithelial cells at the air liquid interphase and a murine model challenged with IAV. The data indicates ESE primarily interacts directly with virions, preventing virus cell binding. Interestingly, ESE also inhibits early and late stage of the influenza A lifecycle when treatment occurs after cell binding. This inhibitory effect appears to prevent internalisation of virus and release of progeny virus by targeting neuraminidase activity. Intranasal administration of ESE in mice infected with IAV reduced viral load in lung tissue. ESE may be a promising broad acting antiviral agent in the treatment of influenza infections.","PeriodicalId":505198,"journal":{"name":"bioRxiv","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141929318","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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