In a lung squamous cell carcinoma data set containing over 20 million tandem mass spectra, we identified 860 peptides with post-translational modifications (PTMs) that were significantly upregulated in lung cancer samples as compared to normal samples using our new search engine named PIPI3. Among the modified peptides related to upregulated gene ontology terms, about 50% carried multiple PTMs. PIPI3 demonstrated its enabling power to provide insight into PTM crosstalk research.
{"title":"Identifying crosstalks among post-translational modifications in lung cancer proteomic data","authors":"Shengzhi Lai, Shuaijian Dai, Peize Zhao, Chen Zhou, Ning Li, Weichuan Yu","doi":"10.1101/2024.08.06.606765","DOIUrl":"https://doi.org/10.1101/2024.08.06.606765","url":null,"abstract":"In a lung squamous cell carcinoma data set containing over 20 million tandem mass spectra, we identified 860 peptides with post-translational modifications (PTMs) that were significantly upregulated in lung cancer samples as compared to normal samples using our new search engine named PIPI3. Among the modified peptides related to upregulated gene ontology terms, about 50% carried multiple PTMs. PIPI3 demonstrated its enabling power to provide insight into PTM crosstalk 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":"141929377","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: 10.1101/2024.08.07.607117
Sara Carrillo Roas, Yuichi Yagita, Paul Murphy, R. Kurzbauer, Tim Clausen, Eszter Zavodszky, R. Hegde
Unassembled and partially assembled subunits of multi-protein complexes have emerged as major quality control clients, particularly under conditions of imbalanced gene expression such as stress, aging, and aneuploidy. The factors and mechanisms that eliminate such orphan subunits to maintain protein homeostasis are incompletely defined. Here, we show that the UBR4-KCMF1 ubiquitin ligase complex is required for efficient degradation of multiple unrelated orphan subunits from the chaperonin, proteasome cap, proteasome core, and a protein targeting complex. Epistasis analysis in cells and reconstitution studies in vitro show that the UBR4-KCMF1 complex acts downstream of a priming ubiquitin ligase that first mono-ubiquitinates orphans. UBR4 recognizes both the orphan and its mono-ubiquitin and builds a K48-linked poly-ubiquitin degradation signal. The discovery of a convergence point for multiple quality control pathways may explain why aneuploid cells are especially sensitive to loss of UBR4 or KCMF1 and identifies a potential vulnerability across many cancers.
{"title":"Convergence of orphan quality control pathways at a ubiquitin chain-elongating ligase","authors":"Sara Carrillo Roas, Yuichi Yagita, Paul Murphy, R. Kurzbauer, Tim Clausen, Eszter Zavodszky, R. Hegde","doi":"10.1101/2024.08.07.607117","DOIUrl":"https://doi.org/10.1101/2024.08.07.607117","url":null,"abstract":"Unassembled and partially assembled subunits of multi-protein complexes have emerged as major quality control clients, particularly under conditions of imbalanced gene expression such as stress, aging, and aneuploidy. The factors and mechanisms that eliminate such orphan subunits to maintain protein homeostasis are incompletely defined. Here, we show that the UBR4-KCMF1 ubiquitin ligase complex is required for efficient degradation of multiple unrelated orphan subunits from the chaperonin, proteasome cap, proteasome core, and a protein targeting complex. Epistasis analysis in cells and reconstitution studies in vitro show that the UBR4-KCMF1 complex acts downstream of a priming ubiquitin ligase that first mono-ubiquitinates orphans. UBR4 recognizes both the orphan and its mono-ubiquitin and builds a K48-linked poly-ubiquitin degradation signal. The discovery of a convergence point for multiple quality control pathways may explain why aneuploid cells are especially sensitive to loss of UBR4 or KCMF1 and identifies a potential vulnerability across many cancers.","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":"141929186","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: 10.1101/2024.08.06.606773
S. Ugwuanyi, M. Makhoul, A. Golicz, C. Obermeier, Rod J. Snowdon
Faba bean (Vicia faba) is a valuable legume crop desired globally for its high nutritional composition. However, the seed vicine and convicine (v-c) content reduces the nutritional quality of faba bean protein and can induce favism in individuals with glucose-6-phosphate dehydrogenase deficiency. Recently, VC1 gene, encoding a bi-functional riboflavin protein, was reported to be responsible for initiating the biosynthetic pathway in V. faba. In low v-c cultivars, a 2 bp insertion in this gene results in a loss of function, but the mutation only partially eliminates v-c biosynthesis, indicating the involvement of other genes. Here, we demonstrate that a novel V. faba riboflavin gene, VC2, is responsible for the residual v-c contents in faba bean. VC2 shares nearly identical functional domains with VC1 and has GTP cyclohydrolase II activity, catalyzing the conversion of GTP into an intermediate molecule in the biosynthetic pathway. Gene expression analysis reveals that VC2 contributes a minor effect to the trait, accounting for approximately 5-10% of total riboflavin gene transcripts which significantly correlates with the baseline contents in low v-c cultivars. Our results illustrate that cultivars carrying the 2 bp inactivating insertion in VC1 still have residual v-c levels due to VC2 activity. Furthermore, we find that VC1 has multiple alleles and exhibits copy number variations, complicating molecular marker development. Conversely, single nucleotide polymorphisms within VC2 provide a reliable alternative for marker-assisted selection in faba bean breeding. In conclusion, our study elucidates the complex genetic regulation of v-c biosynthesis and provides valuable insights to facilitate its elimination in faba bean.
{"title":"VC2 regulates baseline vicine content in faba bean","authors":"S. Ugwuanyi, M. Makhoul, A. Golicz, C. Obermeier, Rod J. Snowdon","doi":"10.1101/2024.08.06.606773","DOIUrl":"https://doi.org/10.1101/2024.08.06.606773","url":null,"abstract":"Faba bean (Vicia faba) is a valuable legume crop desired globally for its high nutritional composition. However, the seed vicine and convicine (v-c) content reduces the nutritional quality of faba bean protein and can induce favism in individuals with glucose-6-phosphate dehydrogenase deficiency. Recently, VC1 gene, encoding a bi-functional riboflavin protein, was reported to be responsible for initiating the biosynthetic pathway in V. faba. In low v-c cultivars, a 2 bp insertion in this gene results in a loss of function, but the mutation only partially eliminates v-c biosynthesis, indicating the involvement of other genes. Here, we demonstrate that a novel V. faba riboflavin gene, VC2, is responsible for the residual v-c contents in faba bean. VC2 shares nearly identical functional domains with VC1 and has GTP cyclohydrolase II activity, catalyzing the conversion of GTP into an intermediate molecule in the biosynthetic pathway. Gene expression analysis reveals that VC2 contributes a minor effect to the trait, accounting for approximately 5-10% of total riboflavin gene transcripts which significantly correlates with the baseline contents in low v-c cultivars. Our results illustrate that cultivars carrying the 2 bp inactivating insertion in VC1 still have residual v-c levels due to VC2 activity. Furthermore, we find that VC1 has multiple alleles and exhibits copy number variations, complicating molecular marker development. Conversely, single nucleotide polymorphisms within VC2 provide a reliable alternative for marker-assisted selection in faba bean breeding. In conclusion, our study elucidates the complex genetic regulation of v-c biosynthesis and provides valuable insights to facilitate its elimination in faba bean.","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":"141928856","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: 10.1101/2024.08.08.607240
Tatiana Demina, Heli Marttila, I. Pessi, Minna K. Männistö, B. Dutilh, Simon Roux, J. Hultman
Acidobacteriota are phylogenetically and physiologically diverse soil bacteria that play important roles in key ecological processes. Still, surprisingly little is known about their viruses. Here, we report five virus isolates, Tunturi 1-5, that were obtained from Arctic tundra soils, Kilpisjärvi, Finland (69°N), using Tunturibacter spp. strains as hosts. The new virus isolates have tailed particles with podo- (Tunturi 1, 2, 3), sipho- (Tunturi 4), or myovirus-like (Tunturi 5) morphologies. The dsDNA genomes of the viral isolates are 63–98 kbp long, except Tunturi 5, which is a jumbo phage with a 309-kbp genome. Tunturi 1 and Tunturi 2 share 88% overall nucleotide identity, while the other three are not related to one another. Over half of the open reading frames in Tunturi genomes have no homologs in the NCBI nr database. Viral diversity in Kilpisjärvi soils was further assessed using a metagenomic approach. A total of 1938 viral operational taxonomic units (vOTUs) were extracted from bulk soil metagenomes, of which 46 represented >90% complete virus genomes. Almost all vOTUs (98%) were assigned to the class Caudoviricetes. For 338 vOTUs, putative hosts were predicted, including Acidobacteriota and other common soil phyla involved in carbon and nitrogen cycling. We also observed 16 putative Terriglobia-associated proviruses in Kilpisjärvi soils, being related to proviruses originating from soils and other distant biomes. Approximately genus- or higher-level similarities could be observed between Tunturi viruses, Kilpisjärvi vOTUs, and other soil vOTUs, suggesting some shared viral diversity across soils. On a local scale, viral communities, including acidobacterial vOTUs, were habitat-specific, being driven by the same environmental factors as their host communities: soil moisture, SOM, C, N content and C:N ratio. This study represents a comprehensive analysis of Acidobacteriota-associated viruses residing in Arctic tundra soils, providing isolates as laboratory models for future studies and adding insights into the viral diversity and virus-host interactions in these climate-critical soils.
{"title":"Tunturi virus isolates and metagenome-assembled viral genomes provide insights into the virome of Acidobacteriota in Arctic tundra soils","authors":"Tatiana Demina, Heli Marttila, I. Pessi, Minna K. Männistö, B. Dutilh, Simon Roux, J. Hultman","doi":"10.1101/2024.08.08.607240","DOIUrl":"https://doi.org/10.1101/2024.08.08.607240","url":null,"abstract":"Acidobacteriota are phylogenetically and physiologically diverse soil bacteria that play important roles in key ecological processes. Still, surprisingly little is known about their viruses. Here, we report five virus isolates, Tunturi 1-5, that were obtained from Arctic tundra soils, Kilpisjärvi, Finland (69°N), using Tunturibacter spp. strains as hosts. The new virus isolates have tailed particles with podo- (Tunturi 1, 2, 3), sipho- (Tunturi 4), or myovirus-like (Tunturi 5) morphologies. The dsDNA genomes of the viral isolates are 63–98 kbp long, except Tunturi 5, which is a jumbo phage with a 309-kbp genome. Tunturi 1 and Tunturi 2 share 88% overall nucleotide identity, while the other three are not related to one another. Over half of the open reading frames in Tunturi genomes have no homologs in the NCBI nr database. Viral diversity in Kilpisjärvi soils was further assessed using a metagenomic approach. A total of 1938 viral operational taxonomic units (vOTUs) were extracted from bulk soil metagenomes, of which 46 represented >90% complete virus genomes. Almost all vOTUs (98%) were assigned to the class Caudoviricetes. For 338 vOTUs, putative hosts were predicted, including Acidobacteriota and other common soil phyla involved in carbon and nitrogen cycling. We also observed 16 putative Terriglobia-associated proviruses in Kilpisjärvi soils, being related to proviruses originating from soils and other distant biomes. Approximately genus- or higher-level similarities could be observed between Tunturi viruses, Kilpisjärvi vOTUs, and other soil vOTUs, suggesting some shared viral diversity across soils. On a local scale, viral communities, including acidobacterial vOTUs, were habitat-specific, being driven by the same environmental factors as their host communities: soil moisture, SOM, C, N content and C:N ratio. This study represents a comprehensive analysis of Acidobacteriota-associated viruses residing in Arctic tundra soils, providing isolates as laboratory models for future studies and adding insights into the viral diversity and virus-host interactions in these climate-critical soils.","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":"141928843","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: 10.1101/2024.08.06.606795
Mosi Li, Kris Holt, Katherine Ridley, Jing Qiu, Kirsty Haddow, Deepali Vasoya, Xin He, J. Tulloch, Declan King, David A. Hume, Clare Pridans, O. Dando, Tara L Spires-Jones, Giles E. Hardingham
Evidence points to a role for microglia in Alzheimer’s disease (AD) risk, although their position in the pathological cascade is incompletely understood, prompting us to generate a model of ß-amyloidopathy lacking microglia. We find evidence that microglia promote plaque formation and creation of an Aß fibril-rich zone surrounding the plaque core. However, plaque-proximal reactive astrogliosis, synapse loss, and neurite dystrophy are still observed in the absence of microglia.
{"title":"Microglia determine ß-amyloid plaque burden but are non-essential for downstream pathology","authors":"Mosi Li, Kris Holt, Katherine Ridley, Jing Qiu, Kirsty Haddow, Deepali Vasoya, Xin He, J. Tulloch, Declan King, David A. Hume, Clare Pridans, O. Dando, Tara L Spires-Jones, Giles E. Hardingham","doi":"10.1101/2024.08.06.606795","DOIUrl":"https://doi.org/10.1101/2024.08.06.606795","url":null,"abstract":"Evidence points to a role for microglia in Alzheimer’s disease (AD) risk, although their position in the pathological cascade is incompletely understood, prompting us to generate a model of ß-amyloidopathy lacking microglia. We find evidence that microglia promote plaque formation and creation of an Aß fibril-rich zone surrounding the plaque core. However, plaque-proximal reactive astrogliosis, synapse loss, and neurite dystrophy are still observed in the absence of microglia.","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":"141926369","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: 10.1101/2024.08.06.606712
Abdulraouf Abdulraouf, Weirong Jiang, Zihan Xu, Zehao Zhang, Samuel Isakov, Tanvir Raihan, Wei Zhou, Junyue Cao
Spatial transcriptomics has revolutionized our understanding of cellular network dynamics in aging and disease by enabling the mapping of molecular and cellular organization across various anatomical locations. Despite these advances, current methods face significant challenges in throughput and cost, limiting their utility for comprehensive studies. To address these limitations, we introduce IRISeq (Imaging Reconstruction using Indexed Sequencing), a optics-free spatial transcriptomics platform that eliminates the need for predefined capture arrays or extensive imaging, allowing for the rapid and cost-effective processing of multiple tissue sections simultaneously. Its capacity to reconstruct images based solely on sequencing local DNA interactions allows for profiling of tissues without size constraints and across varied resolutions. Applying IRISeq, we examined gene expression and cellular dynamics in thirty brain regions of both adult and aged mice, uncovering region-specific changes in gene expression associated with aging. Further cell type-centric analysis further identified age-related cell subtypes and intricate changes in cell interactions that are distinct to certain spatial niches, emphasizing the unique aspects of aging in different brain regions. The affordability and simplicity of IRISeq position it as a versatile tool for mapping region-specific gene expression and cellular interactions across various biological systems. One Sentence Summary: IRISeq, an innovative optics-free spatial transcriptomics method, uncovers aging-related changes in spatial gene expression and focal cell interactions in brain aging.
空间转录组学通过绘制不同解剖位置的分子和细胞组织图，彻底改变了我们对衰老和疾病中细胞网络动态的理解。尽管取得了这些进展，但目前的方法在通量和成本方面仍面临巨大挑战，限制了它们在综合研究中的应用。为了解决这些局限性，我们推出了 IRISeq（使用索引测序的成像重建），这是一种无光学器件的空间转录组学平台，无需预定义的捕获阵列或大量成像，可同时快速、经济高效地处理多个组织切片。它能仅根据局部 DNA 相互作用的测序结果重建图像，因此可以不受尺寸限制地对不同分辨率的组织进行分析。应用 IRISeq，我们检测了成年小鼠和老年小鼠 30 个脑区的基因表达和细胞动态，发现了与衰老相关的特定区域基因表达变化。以细胞类型为中心的进一步分析进一步确定了与年龄相关的细胞亚型和细胞相互作用的复杂变化，这些变化与特定的空间壁龛不同，强调了不同脑区衰老的独特方面。IRISeq 价格低廉、操作简单，是绘制各种生物系统中特定区域基因表达和细胞相互作用图谱的多功能工具。一句话总结：IRISeq 是一种创新的免光学空间转录组学方法，它揭示了大脑衰老过程中与衰老相关的空间基因表达和病灶细胞相互作用的变化。
{"title":"Optics-free Spatial Genomics for Mapping Mouse Brain Aging","authors":"Abdulraouf Abdulraouf, Weirong Jiang, Zihan Xu, Zehao Zhang, Samuel Isakov, Tanvir Raihan, Wei Zhou, Junyue Cao","doi":"10.1101/2024.08.06.606712","DOIUrl":"https://doi.org/10.1101/2024.08.06.606712","url":null,"abstract":"Spatial transcriptomics has revolutionized our understanding of cellular network dynamics in aging and disease by enabling the mapping of molecular and cellular organization across various anatomical locations. Despite these advances, current methods face significant challenges in throughput and cost, limiting their utility for comprehensive studies. To address these limitations, we introduce IRISeq (Imaging Reconstruction using Indexed Sequencing), a optics-free spatial transcriptomics platform that eliminates the need for predefined capture arrays or extensive imaging, allowing for the rapid and cost-effective processing of multiple tissue sections simultaneously. Its capacity to reconstruct images based solely on sequencing local DNA interactions allows for profiling of tissues without size constraints and across varied resolutions. Applying IRISeq, we examined gene expression and cellular dynamics in thirty brain regions of both adult and aged mice, uncovering region-specific changes in gene expression associated with aging. Further cell type-centric analysis further identified age-related cell subtypes and intricate changes in cell interactions that are distinct to certain spatial niches, emphasizing the unique aspects of aging in different brain regions. The affordability and simplicity of IRISeq position it as a versatile tool for mapping region-specific gene expression and cellular interactions across various biological systems. One Sentence Summary: IRISeq, an innovative optics-free spatial transcriptomics method, uncovers aging-related changes in spatial gene expression and focal cell interactions in brain aging.","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":"141927965","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: 10.1101/2024.08.08.607188
Johanna Heid, Zhenqiu Huang, Moonsook Lee, Sergey Makhortov, Elizabeth Pan, Cristina Montagna, Shixiang Sun, Jan Vijg, A. Maslov
Detecting somatic mutations in normal cells and tissues is notoriously challenging due to their low abundance, orders of magnitude below the sequencing error rate. While several techniques, such as single-cell and single-molecule sequencing, have been developed to identify somatic mutations, they are insufficient for detecting genomic structural variants (SVs), which have a significantly greater impact than single-nucleotide variants (SNVs). We introduce Single-Molecule Mutation Sequencing for Structural Variants (SMM-SV-seq), a novel method combining Tn5-mediated, chimera-free library preparation with the precision of error-corrected next-generation sequencing (ecNGS). This approach enhances SV detection accuracy without relying on independent supporting sequencing reads. Our validation studies on human primary fibroblasts treated with varying concentrations of the clastogen bleomycin demonstrated a significant, up to tenfold and dose-dependent, increase in deletions and translocations 24 hours post-treatment. Evaluating SMM-SV-seq’s performance against established computational tools for SV detection, such as Manta and DELLY, using a well-characterized human cell line, SMM-SV-seq showed precision and recall rates of 61.9% and 85.8%, respectively, significantly outperforming Manta (10% precision, 23% recall) and DELLY (15% precision, 32% recall). Using SMM-SV-seq, we documented clear, direct evidence of negative selection against structural variants over time. After a single 2 Gy dose of ionizing radiation, SVs in normal human primary fibroblasts peaked at 24 hours post-intervention and then declined to nearly background levels by day six, highlighting the cellular mechanisms that selectively disadvantage cells harboring these mutations. Additionally, SMM-SV-seq revealed that BRCA1-deficient human breast epithelial cells are more susceptible to the mutagenic effects of ionizing radiation compared to BRCA1-proficient isogenic control cells, suggesting a potential molecular mechanism for increased breast cancer risk in BRCA1 mutation carriers. SMM-SV-seq represents a significant advancement in genomic analysis, enabling the accurate detection of somatic structural variants in normal cells and tissues for the first time. This method complements our previously published Single-Molecule Mutation sequencing (SMM-seq), effective for detecting single-nucleotide variants (SNVs) and small insertions and deletions (INDELs). By addressing challenges such as self-ligation in library preparation and leveraging a powerful ecNGS strategy, SMM-SV-seq enhances the robustness of our genomic analysis toolkit. This breakthrough paves the way for new research into genetic variability and mutation processes, offering deeper insights that could advance our understanding of aging, cancer, and other human diseases.
{"title":"Detection of genome structural variation in normal cells and tissues by single molecule sequencing","authors":"Johanna Heid, Zhenqiu Huang, Moonsook Lee, Sergey Makhortov, Elizabeth Pan, Cristina Montagna, Shixiang Sun, Jan Vijg, A. Maslov","doi":"10.1101/2024.08.08.607188","DOIUrl":"https://doi.org/10.1101/2024.08.08.607188","url":null,"abstract":"Detecting somatic mutations in normal cells and tissues is notoriously challenging due to their low abundance, orders of magnitude below the sequencing error rate. While several techniques, such as single-cell and single-molecule sequencing, have been developed to identify somatic mutations, they are insufficient for detecting genomic structural variants (SVs), which have a significantly greater impact than single-nucleotide variants (SNVs). We introduce Single-Molecule Mutation Sequencing for Structural Variants (SMM-SV-seq), a novel method combining Tn5-mediated, chimera-free library preparation with the precision of error-corrected next-generation sequencing (ecNGS). This approach enhances SV detection accuracy without relying on independent supporting sequencing reads. Our validation studies on human primary fibroblasts treated with varying concentrations of the clastogen bleomycin demonstrated a significant, up to tenfold and dose-dependent, increase in deletions and translocations 24 hours post-treatment. Evaluating SMM-SV-seq’s performance against established computational tools for SV detection, such as Manta and DELLY, using a well-characterized human cell line, SMM-SV-seq showed precision and recall rates of 61.9% and 85.8%, respectively, significantly outperforming Manta (10% precision, 23% recall) and DELLY (15% precision, 32% recall). Using SMM-SV-seq, we documented clear, direct evidence of negative selection against structural variants over time. After a single 2 Gy dose of ionizing radiation, SVs in normal human primary fibroblasts peaked at 24 hours post-intervention and then declined to nearly background levels by day six, highlighting the cellular mechanisms that selectively disadvantage cells harboring these mutations. Additionally, SMM-SV-seq revealed that BRCA1-deficient human breast epithelial cells are more susceptible to the mutagenic effects of ionizing radiation compared to BRCA1-proficient isogenic control cells, suggesting a potential molecular mechanism for increased breast cancer risk in BRCA1 mutation carriers. SMM-SV-seq represents a significant advancement in genomic analysis, enabling the accurate detection of somatic structural variants in normal cells and tissues for the first time. This method complements our previously published Single-Molecule Mutation sequencing (SMM-seq), effective for detecting single-nucleotide variants (SNVs) and small insertions and deletions (INDELs). By addressing challenges such as self-ligation in library preparation and leveraging a powerful ecNGS strategy, SMM-SV-seq enhances the robustness of our genomic analysis toolkit. This breakthrough paves the way for new research into genetic variability and mutation processes, offering deeper insights that could advance our understanding of aging, cancer, and other human 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":"141927047","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: 10.1101/2024.08.06.606898
Sophia Snipes
High-frequency brain oscillations in humans are currently categorized into beta (13-30 Hz) and gamma (>30 Hz). Here, I introduce a new class of oscillations between 25 and 35 Hz, which I propose to call “iota.” Iota oscillations have low amplitudes but can still be measured with surface electroencephalography (EEG). Within an individual, iota has a narrow spectral bandwidth of 2-4 Hz, thus distinguishing it from broadband beta and gamma. Iota oscillations occur as sustained bursts during both wakefulness and REM sleep but do not appear during NREM sleep. They are only found in a minority of individuals, more in children than in adults. Overall, iota oscillations are challenging to detect but could serve as a marker of both brain development and states of vigilance.
{"title":"Iota oscillations (25-35 Hz) during wake and REM sleep in children and young adults","authors":"Sophia Snipes","doi":"10.1101/2024.08.06.606898","DOIUrl":"https://doi.org/10.1101/2024.08.06.606898","url":null,"abstract":"High-frequency brain oscillations in humans are currently categorized into beta (13-30 Hz) and gamma (>30 Hz). Here, I introduce a new class of oscillations between 25 and 35 Hz, which I propose to call “iota.” Iota oscillations have low amplitudes but can still be measured with surface electroencephalography (EEG). Within an individual, iota has a narrow spectral bandwidth of 2-4 Hz, thus distinguishing it from broadband beta and gamma. Iota oscillations occur as sustained bursts during both wakefulness and REM sleep but do not appear during NREM sleep. They are only found in a minority of individuals, more in children than in adults. Overall, iota oscillations are challenging to detect but could serve as a marker of both brain development and states of vigilance.","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":"141928722","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: 10.1101/2024.08.06.606809
Tomasz Slezak, Kelly M. O’Leary, Jinyang Li, A. Rohaim, Elena K. Davydova, A. Kossiakoff
We have developed a portfolio of antibody-based modules that can be prefabricated as standalone units and snapped together in plug-and-play fashion to create uniquely powerful multifunctional assemblies. The basic building blocks are derived from multiple pairs of native and modified Fab scaffolds and protein G (PG) variants engineered by phage display to introduce high pair-wise specificity. The variety of possible Fab-PG pairings provides a highly orthogonal system that can be exploited to perform challenging cell biology operations in a straightforward manner. The simplest manifestation allows multiplexed antigen detection using PG variants fused to fluorescently labeled SNAP-tags. Moreover, Fabs can be readily attached to a PG-Fc dimer module which acts as the core unit to produce plug-and-play IgG-like assemblies, and the utility can be further expanded to produce bispecific analogs using the “knobs into holes” strategy. These core PG-Fc dimer modules can be made and stored in bulk to produce off-the-shelf customized IgG entities in minutes, not days or weeks by just adding a Fab with the desired antigen specificity. In another application, the bispecific modalities form the building block for fabricating potent Bispecific T-cell Engagers (BiTEs), demonstrating their efficacy in cancer cell-killing assays. Additionally, the system can be adapted to include commercial antibodies as building blocks, greatly increasing the target space. Crystal structure analysis reveals that a few strategically positioned interactions engender the specificity between the Fab-PG variant pairs, requiring minimal changes to match the scaffolds for different possible combinations. This plug-and-play platform offers a user-friendly and versatile approach to enhance the functionality of antibody-based reagents in cell biology research.
我们已开发出一系列基于抗体的模块，这些模块可作为独立单元预制，并以即插即用的方式拼接在一起，形成功能独特强大的多功能组件。这些基本构件来自多对原生和修饰的 Fab 支架和蛋白 G (PG) 变体，这些变体通过噬菌体展示工程引入了高配对特异性。各种可能的 Fab-PG 配对提供了一个高度正交的系统，可用于以简单的方式执行具有挑战性的细胞生物学操作。最简单的表现形式是使用融合了荧光标记 SNAP 标签的 PG 变体进行多重抗原检测。此外，Fabs 可以很容易地连接到 PG-Fc 二聚体模块上，而 PG-Fc 二聚体模块是生产即插即用类 IgG 组合物的核心单元，其用途还可以进一步扩展，利用 "钮入孔 "策略生产双特异性类似物。这些核心 PG-Fc 二聚体模块可以批量生产和储存，只需添加具有所需抗原特异性的 Fab，就能在几分钟内而不是几天或几周内生产出现成的定制 IgG 实体。在另一项应用中，双特异性模式构成了制造强效双特异性 T 细胞激活剂（BiTE）的基石，在癌细胞杀伤试验中证明了它们的功效。此外，该系统还可以将商业抗体作为构建模块，从而大大增加了靶标空间。晶体结构分析表明，Fab-PG 变体对之间的特异性是由几个策略性定位的相互作用产生的，只需做极少的改动就能匹配不同可能组合的支架。这种即插即用的平台为增强细胞生物学研究中基于抗体的试剂的功能提供了一种用户友好型多功能方法。
{"title":"Engineered Protein-G variants for plug-and-play applications","authors":"Tomasz Slezak, Kelly M. O’Leary, Jinyang Li, A. Rohaim, Elena K. Davydova, A. Kossiakoff","doi":"10.1101/2024.08.06.606809","DOIUrl":"https://doi.org/10.1101/2024.08.06.606809","url":null,"abstract":"We have developed a portfolio of antibody-based modules that can be prefabricated as standalone units and snapped together in plug-and-play fashion to create uniquely powerful multifunctional assemblies. The basic building blocks are derived from multiple pairs of native and modified Fab scaffolds and protein G (PG) variants engineered by phage display to introduce high pair-wise specificity. The variety of possible Fab-PG pairings provides a highly orthogonal system that can be exploited to perform challenging cell biology operations in a straightforward manner. The simplest manifestation allows multiplexed antigen detection using PG variants fused to fluorescently labeled SNAP-tags. Moreover, Fabs can be readily attached to a PG-Fc dimer module which acts as the core unit to produce plug-and-play IgG-like assemblies, and the utility can be further expanded to produce bispecific analogs using the “knobs into holes” strategy. These core PG-Fc dimer modules can be made and stored in bulk to produce off-the-shelf customized IgG entities in minutes, not days or weeks by just adding a Fab with the desired antigen specificity. In another application, the bispecific modalities form the building block for fabricating potent Bispecific T-cell Engagers (BiTEs), demonstrating their efficacy in cancer cell-killing assays. Additionally, the system can be adapted to include commercial antibodies as building blocks, greatly increasing the target space. Crystal structure analysis reveals that a few strategically positioned interactions engender the specificity between the Fab-PG variant pairs, requiring minimal changes to match the scaffolds for different possible combinations. This plug-and-play platform offers a user-friendly and versatile approach to enhance the functionality of antibody-based reagents in cell biology 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":"141926320","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: 10.1101/2024.08.08.607148
Yizhuo Wang, Hengfang Tang, Wanxue Wang, Ming Li, Chenchen Zhu, Han Dai, Hongxin Zhao, Bo Wu, Junfeng Wang
Variant surface antigens of Plasmodium falciparum, including RIFIN, play a pivotal role in malaria pathogenesis and facilitate immune evasion by binding to immunoinhibitory receptors such as LILRB1. Recently, receptor-containing antibodies have been discovered in malaria-exposed individuals and uncover a novel antibody mechanism in inhibiting immune evasions of Plasmodium falciparum. Previous studies have identified several LAIR1– and LILRB1 D3D4-containing antibodies. However, no antibodies containing LILRB1-D1D2 have been identified, even though some RIFINs interact with LILRB1-D1D2. In this study, we propose a in vitro strategy for the generation of this type of antibodies by employing structure-based affinity maturation. Using this strategy, we successfully generated D1D2.v-IgG, an antibody that effectively blocks the specific binding of RIFIN#1 (from PF3D7_1254800) to LILRB1. Furthermore, we developed NK-biAb, a bispecific antibody targeting RIFIN#1 and the NKG2D receptor based on D1D2.v-IgG. Both antibodies demonstrate promising results in augmenting NK cell-mediated cytotoxicity against RIFIN#1-expressing K562 cells, with NK-biAb exhibiting superior efficacy. The present strategy could be generally used for developing antibodies against the malarial parasite-host interactions, thereby facilitating advancements in malaria treatments and vaccines.
{"title":"Potent LILRB1 D1D2-containing antibodies inhibit RIFIN-mediated immune evasions","authors":"Yizhuo Wang, Hengfang Tang, Wanxue Wang, Ming Li, Chenchen Zhu, Han Dai, Hongxin Zhao, Bo Wu, Junfeng Wang","doi":"10.1101/2024.08.08.607148","DOIUrl":"https://doi.org/10.1101/2024.08.08.607148","url":null,"abstract":"Variant surface antigens of Plasmodium falciparum, including RIFIN, play a pivotal role in malaria pathogenesis and facilitate immune evasion by binding to immunoinhibitory receptors such as LILRB1. Recently, receptor-containing antibodies have been discovered in malaria-exposed individuals and uncover a novel antibody mechanism in inhibiting immune evasions of Plasmodium falciparum. Previous studies have identified several LAIR1– and LILRB1 D3D4-containing antibodies. However, no antibodies containing LILRB1-D1D2 have been identified, even though some RIFINs interact with LILRB1-D1D2. In this study, we propose a in vitro strategy for the generation of this type of antibodies by employing structure-based affinity maturation. Using this strategy, we successfully generated D1D2.v-IgG, an antibody that effectively blocks the specific binding of RIFIN#1 (from PF3D7_1254800) to LILRB1. Furthermore, we developed NK-biAb, a bispecific antibody targeting RIFIN#1 and the NKG2D receptor based on D1D2.v-IgG. Both antibodies demonstrate promising results in augmenting NK cell-mediated cytotoxicity against RIFIN#1-expressing K562 cells, with NK-biAb exhibiting superior efficacy. The present strategy could be generally used for developing antibodies against the malarial parasite-host interactions, thereby facilitating advancements in malaria treatments and vaccines.","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":"141929122","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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