A novel fluorogenic reporter substrate for 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2): Application to high-throughput screening for activators to treat Alzheimer's disease Visvanathan, Ramya In: 2023. @article{noKey,
title = {A novel fluorogenic reporter substrate for 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2): Application to high-throughput screening for activators to treat Alzheimer's disease},
author = {Visvanathan, Ramya},
url = {https://slas-discovery.org/article/S2472-5552(23)00024-2/fulltext},
doi = {https://doi.org/10.1016/j.slasd.2023.03.003},
year = {2023},
date = {2023-01-01},
abstract = {A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various “turn-on” fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various “turn-on” fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening. |
Purification and characterization of human neural stem and progenitor cells Liu, Daniel Dan In: 2023. @article{noKey,
title = {Purification and characterization of human neural stem and progenitor cells},
author = {Liu, Daniel Dan},
url = {https://www.cell.com/cell/fulltext/S0092-8674(23)00160-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867423001605%3Fshowall%3Dtrue},
doi = {https://doi.org/10.1016/j.cell.2023.02.017},
year = {2023},
date = {2023-01-01},
abstract = {The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24−THY1−/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1−/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA− bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24−THY1−/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1−/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA− bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment. |
Core species and interactions prominent in fish-associated microbiome dynamics Yajima, Daii In: 2023. @article{noKey,
title = {Core species and interactions prominent in fish-associated microbiome dynamics},
author = {Yajima, Daii},
url = {https://link.springer.com/article/10.1186/s40168-023-01498-x},
doi = {https://doi.org/10.1186/s40168-023-01498-x},
year = {2023},
date = {2023-01-01},
abstract = {Background
In aquatic ecosystems, the health and performance of fish depend greatly on the dynamics of microbial community structure in the background environment. Nonetheless, finding microbes with profound impacts on fish’s performance out of thousands of candidate species remains a major challenge.
Methods
We examined whether time-series analyses of microbial population dynamics could illuminate core components and structure of fish-associated microbiomes in the background (environmental) water. By targeting eel-aquaculture-tank microbiomes as model systems, we reconstructed the population dynamics of the 9605 bacterial and 303 archaeal species/strains across 128 days.
Results
Due to the remarkable increase/decrease of constituent microbial population densities, the taxonomic compositions of the microbiome changed drastically through time. We then found that some specific microbial taxa showed a positive relationship with eels’ activity levels even after excluding confounding effects of environmental parameters (pH and dissolved oxygen level) on population dynamics. In particular, a vitamin-B12-producing bacteria, Cetobacterium somerae, consistently showed strong positive associations with eels’ activity levels across the replicate time series of the five aquaculture tanks analyzed. Network theoretical and metabolic modeling analyses further suggested that the highlighted bacterium and some other closely-associated bacteria formed “core microbiomes” with potentially positive impacts on eels.
Conclusions
Overall, these results suggest that the integration of microbiology, ecological theory, and network science allows us to explore core species and interactions embedded within complex dynamics of fish-associated microbiomes.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Background
In aquatic ecosystems, the health and performance of fish depend greatly on the dynamics of microbial community structure in the background environment. Nonetheless, finding microbes with profound impacts on fish’s performance out of thousands of candidate species remains a major challenge.
Methods
We examined whether time-series analyses of microbial population dynamics could illuminate core components and structure of fish-associated microbiomes in the background (environmental) water. By targeting eel-aquaculture-tank microbiomes as model systems, we reconstructed the population dynamics of the 9605 bacterial and 303 archaeal species/strains across 128 days.
Results
Due to the remarkable increase/decrease of constituent microbial population densities, the taxonomic compositions of the microbiome changed drastically through time. We then found that some specific microbial taxa showed a positive relationship with eels’ activity levels even after excluding confounding effects of environmental parameters (pH and dissolved oxygen level) on population dynamics. In particular, a vitamin-B12-producing bacteria, Cetobacterium somerae, consistently showed strong positive associations with eels’ activity levels across the replicate time series of the five aquaculture tanks analyzed. Network theoretical and metabolic modeling analyses further suggested that the highlighted bacterium and some other closely-associated bacteria formed “core microbiomes” with potentially positive impacts on eels.
Conclusions
Overall, these results suggest that the integration of microbiology, ecological theory, and network science allows us to explore core species and interactions embedded within complex dynamics of fish-associated microbiomes. |
Isolation and characterisation of novel Methanocorpusculum species indicates the genus is ancestrally host-associated Volmer, James G. In: 2023. @article{noKey,
title = {Isolation and characterisation of novel Methanocorpusculum species indicates the genus is ancestrally host-associated},
author = {Volmer, James G.},
url = {https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-023-01524-2},
doi = {https://doi.org/10.1186/s12915-023-01524-2},
year = {2023},
date = {2023-01-01},
abstract = {Background
With an increasing interest in the manipulation of methane produced from livestock cultivation, the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with ‘low-methane’ emitters. Previously, marsupial species were shown to be enriched for novel lineages of Methanocorpusculum, as well as Methanobrevibacter, Methanosphaera, and Methanomassiliicoccales. Despite sporadic reports of Methanocorpusculum from stool samples of various animal species, there remains little information on the impacts of these methanogens on their hosts.
Results
Here, we characterise novel host-associated species of Methanocorpusculum, to explore unique host-specific genetic factors and their associated metabolic potential. We performed comparative analyses on 176 Methanocorpusculum genomes comprising 130 metagenome-assembled genomes (MAGs) recovered from 20 public animal metagenome datasets and 35 other publicly available Methanocorpusculum MAGs and isolate genomes of host-associated and environmental origin. Nine MAGs were also produced from faecal metagenomes of the common wombat (Vombatus ursinus) and mahogany glider (Petaurus gracilis), along with the cultivation of one axenic isolate from each respective animal; M. vombati (sp. nov.) and M. petauri (sp. nov.).
Conclusions
Through our analyses, we substantially expand the available genetic information for this genus by describing the phenotypic and genetic characteristics of 23 host-associated species of Methanocorpusculum. These lineages display differential enrichment of genes associated with methanogenesis, amino acid biosynthesis, transport system proteins, phosphonate metabolism, and carbohydrate-active enzymes. These results provide insights into the differential genetic and functional adaptations of these novel host-associated species of Methanocorpusculum and suggest that this genus is ancestrally host-associated.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Background
With an increasing interest in the manipulation of methane produced from livestock cultivation, the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with ‘low-methane’ emitters. Previously, marsupial species were shown to be enriched for novel lineages of Methanocorpusculum, as well as Methanobrevibacter, Methanosphaera, and Methanomassiliicoccales. Despite sporadic reports of Methanocorpusculum from stool samples of various animal species, there remains little information on the impacts of these methanogens on their hosts.
Results
Here, we characterise novel host-associated species of Methanocorpusculum, to explore unique host-specific genetic factors and their associated metabolic potential. We performed comparative analyses on 176 Methanocorpusculum genomes comprising 130 metagenome-assembled genomes (MAGs) recovered from 20 public animal metagenome datasets and 35 other publicly available Methanocorpusculum MAGs and isolate genomes of host-associated and environmental origin. Nine MAGs were also produced from faecal metagenomes of the common wombat (Vombatus ursinus) and mahogany glider (Petaurus gracilis), along with the cultivation of one axenic isolate from each respective animal; M. vombati (sp. nov.) and M. petauri (sp. nov.).
Conclusions
Through our analyses, we substantially expand the available genetic information for this genus by describing the phenotypic and genetic characteristics of 23 host-associated species of Methanocorpusculum. These lineages display differential enrichment of genes associated with methanogenesis, amino acid biosynthesis, transport system proteins, phosphonate metabolism, and carbohydrate-active enzymes. These results provide insights into the differential genetic and functional adaptations of these novel host-associated species of Methanocorpusculum and suggest that this genus is ancestrally host-associated. |
Phylogenetic and metabolic diversity of microbial communities performing anaerobic ammonium and methane oxidations under different nitrogen loadings Li, Jie In: 2023. @article{noKey,
title = {Phylogenetic and metabolic diversity of microbial communities performing anaerobic ammonium and methane oxidations under different nitrogen loadings},
author = {Li, Jie},
url = {https://www.nature.com/articles/s43705-023-00246-4#Sec19},
doi = {https://doi.org/10.1038/s43705-023-00246-4},
year = {2023},
date = {2023-01-01},
abstract = {The microbial guild coupling anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater. Here, metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems, which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater, respectively. We find that different nitrogen loadings (i.e., 0.1 vs. 1.0 kg N/m3/d) lead to different combinations of anammox bacteria and anaerobic methanotrophs (“Candidatus Methanoperedens” and “Candidatus Methylomirabilis”), which play primary roles for carbon and nitrogen transformations therein. Despite methane being the only exogenous organic carbon supplied, heterotrophic populations (e.g., Verrucomicrobiota and Bacteroidota) co-exist and actively perform partial denitrification or dissimilatory nitrate reduction to ammonium (DNRA), likely using organic intermediates from the breakdown of methane and biomass as carbon sources. More importantly, two novel genomes belonging to “Ca. Methylomirabilis” are recovered, while one surprisingly expresses nitrate reductases, which we designate as “Ca. Methylomirabilis nitratireducens” representing its inferred capability in performing nitrate-dependent anaerobic methane oxidation. This finding not only suggests a previously neglected possibility of “Ca. Methylomirabilis” bacteria in performing methane-dependent nitrate reduction, and also challenges the previous understanding that the methane-dependent complete denitrification from nitrate to dinitrogen gas is carried out by the consortium of bacteria and archaea.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The microbial guild coupling anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater. Here, metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems, which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater, respectively. We find that different nitrogen loadings (i.e., 0.1 vs. 1.0 kg N/m3/d) lead to different combinations of anammox bacteria and anaerobic methanotrophs (“Candidatus Methanoperedens” and “Candidatus Methylomirabilis”), which play primary roles for carbon and nitrogen transformations therein. Despite methane being the only exogenous organic carbon supplied, heterotrophic populations (e.g., Verrucomicrobiota and Bacteroidota) co-exist and actively perform partial denitrification or dissimilatory nitrate reduction to ammonium (DNRA), likely using organic intermediates from the breakdown of methane and biomass as carbon sources. More importantly, two novel genomes belonging to “Ca. Methylomirabilis” are recovered, while one surprisingly expresses nitrate reductases, which we designate as “Ca. Methylomirabilis nitratireducens” representing its inferred capability in performing nitrate-dependent anaerobic methane oxidation. This finding not only suggests a previously neglected possibility of “Ca. Methylomirabilis” bacteria in performing methane-dependent nitrate reduction, and also challenges the previous understanding that the methane-dependent complete denitrification from nitrate to dinitrogen gas is carried out by the consortium of bacteria and archaea. |
High-Throughput Screening to Develop a Biologically Relevant Netosis Induction Model Zukas et, Kieran In: 2023. @article{noKey,
title = {High-Throughput Screening to Develop a Biologically Relevant Netosis Induction Model},
author = {Zukas et, Kieran},
url = {https://www.sciencedirect.com/science/article/abs/pii/S000649712310509X},
doi = {https://doi.org/10.1182/blood-2023-178975},
year = {2023},
date = {2023-01-01},
abstract = {Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection (1). It is responsible for ~370,000 US deaths annually (2). Neutrophils are an integral part of the innate immune response and rapidly clear pathogens from circulation using neutrophil extracellular traps (NETs), which are released through a process called NETosis (3). NETs prevent dissemination of pathogens by entrapment in externalized chromatin containing deactivating enzymes. While we have learned much about the mechanisms underlying NETosis, we are yet to translate it to improved therapies or patient outcomes. This gap may be attributable to the models used to study NETosis. Current models used to investigate NETosis are limited and routinely employ unnatural triggers such as phorbol 12-myristate 13-acetate (PMA). PMA is not a physiological trigger present in the immune system and may bypass the natural pathways that regulate NETs production. Mouse models that use isolated neutrophils and neutrophil-like cells induced from immortalized cell lines do not completely reflect the complex cellular and molecular biology underlying neutrophil activation and NETosis, especially in a whole-blood environment. Therefore, it is crucial to study how specific factors, known to be upregulated in disease, interact and potentially induce NETosis. Here we use high-throughput screening and natural NETosis triggers to develop a more biologically relevant ex vivo NETosis (Synthetic-Sepsis™) model.
Whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: Il-1b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 and other molecules TNF- α, LT-α, IFN-γ, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1, CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, Ferritin, HMGB1, C5a and LPS. We used a combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations.
Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and/or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors are necessary to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-α or LT-α was required to cause a NETosis response, underlining the potentially significant roles these factors play in inflammatory disease. These results suggest an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.
To our knowledge, we report the first ex-vivo model using naturally occurring cytokines and chemokines to induce NETosis in whole blood. These findings emphasize the importance of expanding our understanding of neutrophil physiology in a biologically relevant context with physiological triggers to induce NETosis. This approach could reveal new dimensions in our understanding of disease pathology and risk factors and might unearth potential therapeutic targets providing novel strategies for disease intervention and treatment. Further investigation of these factors is underway to further understand the release of NETs in natural and pathological states.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection (1). It is responsible for ~370,000 US deaths annually (2). Neutrophils are an integral part of the innate immune response and rapidly clear pathogens from circulation using neutrophil extracellular traps (NETs), which are released through a process called NETosis (3). NETs prevent dissemination of pathogens by entrapment in externalized chromatin containing deactivating enzymes. While we have learned much about the mechanisms underlying NETosis, we are yet to translate it to improved therapies or patient outcomes. This gap may be attributable to the models used to study NETosis. Current models used to investigate NETosis are limited and routinely employ unnatural triggers such as phorbol 12-myristate 13-acetate (PMA). PMA is not a physiological trigger present in the immune system and may bypass the natural pathways that regulate NETs production. Mouse models that use isolated neutrophils and neutrophil-like cells induced from immortalized cell lines do not completely reflect the complex cellular and molecular biology underlying neutrophil activation and NETosis, especially in a whole-blood environment. Therefore, it is crucial to study how specific factors, known to be upregulated in disease, interact and potentially induce NETosis. Here we use high-throughput screening and natural NETosis triggers to develop a more biologically relevant ex vivo NETosis (Synthetic-Sepsis™) model.
Whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: Il-1b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 and other molecules TNF- α, LT-α, IFN-γ, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1, CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, Ferritin, HMGB1, C5a and LPS. We used a combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations.
Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and/or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors are necessary to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-α or LT-α was required to cause a NETosis response, underlining the potentially significant roles these factors play in inflammatory disease. These results suggest an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.
To our knowledge, we report the first ex-vivo model using naturally occurring cytokines and chemokines to induce NETosis in whole blood. These findings emphasize the importance of expanding our understanding of neutrophil physiology in a biologically relevant context with physiological triggers to induce NETosis. This approach could reveal new dimensions in our understanding of disease pathology and risk factors and might unearth potential therapeutic targets providing novel strategies for disease intervention and treatment. Further investigation of these factors is underway to further understand the release of NETs in natural and pathological states. |
Virtual Screening Assisted Search for Inhibitors of the Translocated Intimin Receptor of Enteropathogenic Escherichia Coli Pylkko et, Tuomas In: 2023. @article{noKey,
title = {Virtual Screening Assisted Search for Inhibitors of the Translocated Intimin Receptor of Enteropathogenic Escherichia Coli},
author = {Pylkko et, Tuomas},
url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cbic.202300638},
doi = {https://doi.org/10.1002/cbic.202300638},
year = {2023},
date = {2023-01-01},
abstract = {This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections. |
Discovery of 2-amide-3-methylester thiophenes inhibiting SARS-CoV-2 ADP-ribosyl hydrolysing macrodomain and coronavirus replication Wazir, Sarah In: 2023. @article{noKey,
title = {Discovery of 2-amide-3-methylester thiophenes inhibiting SARS-CoV-2 ADP-ribosyl hydrolysing macrodomain and coronavirus replication},
author = {Wazir, Sarah},
url = {https://www.biorxiv.org/content/10.1101/2023.08.28.555062v1.full},
doi = {https://doi.org/10.1101/2023.08.28.555062},
year = {2023},
date = {2023-01-01},
abstract = {The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 µM. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of a mouse hepatitis virus, a prototype CoV. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 µM. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of a mouse hepatitis virus, a prototype CoV. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts. |
Skim exome capture genotyping in wheat Wang, Hongliang, In: 2023. @article{noKey,
title = {Skim exome capture genotyping in wheat},
author = {Wang, Hongliang,},
url = {https://acsess.onlinelibrary.wiley.com/doi/10.1002/tpg2.20381},
doi = {https://doi.org/10.1002/tpg2.20381},
year = {2023},
date = {2023-01-01},
abstract = {Next-generation sequencing (NGS) technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research. Skim sequencing, which surveys the entire genome at low coverage, has become feasible for quantitative trait locus (QTL) mapping and genomic selection in various crops. However, the genome complexity of allopolyploid crops such as wheat (Triticum aestivum L.) still poses a significant challenge for genome-wide genotyping. Targeted sequencing of the protein-coding regions (i.e., exome) reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping. We developed a method called skim exome capture (SEC) that combines the strengths of these existing technologies and produces targeted genotyping data while decreasing the cost on a per-sample basis compared to traditional exome capture. Specifically, we fragmented genomic DNA using a tagmentation approach, then enriched those fragments for the low-copy genic portion of the genome using commercial wheat exome baits and multiplexed the sequencing at different levels to achieve desired coverage. We demonstrated that for a library of 48 samples, ∼7–8× target coverage was sufficient for high-quality variant detection. For higher multiplexing levels of 528 and 1056 samples per library, we achieved an average coverage of 0.76× and 0.32×, respectively. Combining these lower coverage SEC sequencing data with genotype imputation using a customized wheat practical haplotype graph database that we developed, we identified hundreds of thousands of high-quality genic variants across the genome. The SEC method can be used for high-resolution QTL mapping, genome-wide association studies, genomic selection, and other downstream applications.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Next-generation sequencing (NGS) technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research. Skim sequencing, which surveys the entire genome at low coverage, has become feasible for quantitative trait locus (QTL) mapping and genomic selection in various crops. However, the genome complexity of allopolyploid crops such as wheat (Triticum aestivum L.) still poses a significant challenge for genome-wide genotyping. Targeted sequencing of the protein-coding regions (i.e., exome) reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping. We developed a method called skim exome capture (SEC) that combines the strengths of these existing technologies and produces targeted genotyping data while decreasing the cost on a per-sample basis compared to traditional exome capture. Specifically, we fragmented genomic DNA using a tagmentation approach, then enriched those fragments for the low-copy genic portion of the genome using commercial wheat exome baits and multiplexed the sequencing at different levels to achieve desired coverage. We demonstrated that for a library of 48 samples, ∼7–8× target coverage was sufficient for high-quality variant detection. For higher multiplexing levels of 528 and 1056 samples per library, we achieved an average coverage of 0.76× and 0.32×, respectively. Combining these lower coverage SEC sequencing data with genotype imputation using a customized wheat practical haplotype graph database that we developed, we identified hundreds of thousands of high-quality genic variants across the genome. The SEC method can be used for high-resolution QTL mapping, genome-wide association studies, genomic selection, and other downstream applications. |
Single Cell Proteomics Analysis of Drug Response Shows its Potential as a Drug Discovery Platform Juerg, Straubhaar, Alexandria, D'Souza In: 2023. @article{noKey,
title = {Single Cell Proteomics Analysis of Drug Response Shows its Potential as a Drug Discovery Platform},
author = {Juerg, Straubhaar, Alexandria, D'Souza},
url = {https://pubs.rsc.org/en/content/articlelanding/2024/MO/D3MO00124E},
doi = {https://doi.org/10.1039/D3MO00124E},
year = {2023},
date = {2023-01-01},
abstract = {Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells, including their types, states, transitions, trajectories, and spatial position. Scientific methods such as fluorescence labeling, nanoscale super-resolution microscopy, advances in single cell RNAseq and proteomics technologies, provide more detailed information about biological processes which were not evident with the analysis of bulk material. This new era of single-cell biology provides a better understanding of such complex biological systems as cancer, inflammation, immunity mechanism and aging processes, and opens the door into the field of drug response heterogeneity. The latest discoveries of cellular heterogeneity gives us an unique understanding of complex biological processes, such as disease mechanism, and will lead to new strategies for better and personalized treatment strategies. Recently, single-cell proteomics techniques that allow quantification of thousands of proteins from single mammalian cells have been introduced. Here we present an improved single-cell mass spectrometry-based proteomics platform called SCREEN (Single Cell pRotEomE aNalysis) for deep and high-throughput single-cell proteome coverage with high efficiency, less turnaround time and with an improved ability for protein quantitation across more cells than previously achieved. We applied this new platform to analyze the single-cell proteomic landscape under different drug treatment over time to uncover heterogeneity in cancer cell response, which for the first time, to our knowledge, has been achieved by mass spectrometry based analytical methods. We discuss challenges in single-cell proteomics, future improvements and general trends with the goal to encourage forthcoming technical developments.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells, including their types, states, transitions, trajectories, and spatial position. Scientific methods such as fluorescence labeling, nanoscale super-resolution microscopy, advances in single cell RNAseq and proteomics technologies, provide more detailed information about biological processes which were not evident with the analysis of bulk material. This new era of single-cell biology provides a better understanding of such complex biological systems as cancer, inflammation, immunity mechanism and aging processes, and opens the door into the field of drug response heterogeneity. The latest discoveries of cellular heterogeneity gives us an unique understanding of complex biological processes, such as disease mechanism, and will lead to new strategies for better and personalized treatment strategies. Recently, single-cell proteomics techniques that allow quantification of thousands of proteins from single mammalian cells have been introduced. Here we present an improved single-cell mass spectrometry-based proteomics platform called SCREEN (Single Cell pRotEomE aNalysis) for deep and high-throughput single-cell proteome coverage with high efficiency, less turnaround time and with an improved ability for protein quantitation across more cells than previously achieved. We applied this new platform to analyze the single-cell proteomic landscape under different drug treatment over time to uncover heterogeneity in cancer cell response, which for the first time, to our knowledge, has been achieved by mass spectrometry based analytical methods. We discuss challenges in single-cell proteomics, future improvements and general trends with the goal to encourage forthcoming technical developments. |
Exploring positions 6 and 7 of a quinazoline-based scaffold leads to changes in selectivity and potency towards RIPK2/3 kinases Misehe, Mbilo In: 2023. @article{noKey,
title = {Exploring positions 6 and 7 of a quinazoline-based scaffold leads to changes in selectivity and potency towards RIPK2/3 kinases},
author = {Misehe, Mbilo},
url = {https://www.sciencedirect.com/science/article/pii/S0223523423006840},
doi = {https://doi.org/10.1016/j.ejmech.2023.115717},
year = {2023},
date = {2023-01-01},
abstract = {Receptor-interacting protein kinases 2 and 3 (RIPK2 and RIPK3) are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers. In this study, we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions 6 and 7 of the central core on the inhibitory activity and the selectivity against these RIPKs. The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK2 and RIPK3 in complex with known quinazoline or quinoline inhibitors. Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs (RIPK1-4) shed light on multiple potent and selective RIPK2 and dual RIPK2/3 inhibitors. Furthermore, evaluations in living cells against the RIPK2-NOD1/2-mediated signaling pathways, identified as the potential primary targets, demonstrated nanomolar inhibition for a majority of the compounds. In addition, we have demonstrated overall good stability of various lead inhibitors in both human and mouse microsomes and plasma. Several of these compounds also were evaluated for selectivity across 58 human kinases other than RIPKs, exhibiting outstanding specificity profiles. We have thus clearly demonstrated that tuning appropriate substitutions at positions 6 and 7 of the developed quinazoline derivatives may lead to interesting potency and specificities against RIPK2 and RIPK3. This knowledge might therefore be employed for the targeted preparation of new, highly potent and selective tools against these RIPKs, which could be of utility in biological and clinical research.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Receptor-interacting protein kinases 2 and 3 (RIPK2 and RIPK3) are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers. In this study, we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions 6 and 7 of the central core on the inhibitory activity and the selectivity against these RIPKs. The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK2 and RIPK3 in complex with known quinazoline or quinoline inhibitors. Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs (RIPK1-4) shed light on multiple potent and selective RIPK2 and dual RIPK2/3 inhibitors. Furthermore, evaluations in living cells against the RIPK2-NOD1/2-mediated signaling pathways, identified as the potential primary targets, demonstrated nanomolar inhibition for a majority of the compounds. In addition, we have demonstrated overall good stability of various lead inhibitors in both human and mouse microsomes and plasma. Several of these compounds also were evaluated for selectivity across 58 human kinases other than RIPKs, exhibiting outstanding specificity profiles. We have thus clearly demonstrated that tuning appropriate substitutions at positions 6 and 7 of the developed quinazoline derivatives may lead to interesting potency and specificities against RIPK2 and RIPK3. This knowledge might therefore be employed for the targeted preparation of new, highly potent and selective tools against these RIPKs, which could be of utility in biological and clinical research. |
Glycidamide-induced hypermutation in yeast single-stranded DNA reveals a ubiquitous clock-like mutational motif in humans Hudson, Kathleen M In: 2023. @article{noKey,
title = {Glycidamide-induced hypermutation in yeast single-stranded DNA reveals a ubiquitous clock-like mutational motif in humans},
author = {Hudson, Kathleen M},
url = {https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkad611/7226869?login=false},
doi = {https://doi.org/10.1093/nar/gkad611},
year = {2023},
date = {2023-01-01},
abstract = {Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents. |
scEC&T-seq: a method for parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single human cells González, Rocío In: 2023. @article{noKey,
title = {scEC&T-seq: a method for parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single human cells},
author = {González, Rocío},
url = {https://europepmc.org/article/ppr/ppr665786},
doi = {https://doi.org/10.21203/rs.3.pex-2180/v1},
year = {2023},
date = {2023-01-01},
abstract = {Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival. A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity. Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy. Here, we introduce scEC&T-seq ( s ingle c ell e xtrachromosomal c ircular DNA and t ranscriptomic seq uencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells. In this protocol, a single cell’s DNA is separated from its polyadenylated RNA as described by Macaulay et al. (2015) 1 . This is followed by removal of linear DNA through exonuclease digestion and further enrichment of circular DNA by rolling circle amplification with φ29 polymerase 2-4 . The separated mRNA from the same cell is processed using on-bead Smart-seq2 1 . The duration of the entire procedure from cell sorting to library preparation is approximately 8 days. Our scEC&T-seq protocol has been validated in single cancer cells from neuroblastoma cell lines and primary tumors, and in normal single T-cells isolated from patient’s blood. Besides identifying large, oncogene-containing circular DNAs in cancer cells, our method also captures other smaller circular DNAs, which have been previously described in both cancer and non-malignant cells 5 . We envision that our method may enable the analysis of yet unknown prerequisites for the maintenance of both small and large circular DNA in cancers, but also in the context of other diseases and normal cellular development.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival. A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity. Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy. Here, we introduce scEC&T-seq ( s ingle c ell e xtrachromosomal c ircular DNA and t ranscriptomic seq uencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells. In this protocol, a single cell’s DNA is separated from its polyadenylated RNA as described by Macaulay et al. (2015) 1 . This is followed by removal of linear DNA through exonuclease digestion and further enrichment of circular DNA by rolling circle amplification with φ29 polymerase 2-4 . The separated mRNA from the same cell is processed using on-bead Smart-seq2 1 . The duration of the entire procedure from cell sorting to library preparation is approximately 8 days. Our scEC&T-seq protocol has been validated in single cancer cells from neuroblastoma cell lines and primary tumors, and in normal single T-cells isolated from patient’s blood. Besides identifying large, oncogene-containing circular DNAs in cancer cells, our method also captures other smaller circular DNAs, which have been previously described in both cancer and non-malignant cells 5 . We envision that our method may enable the analysis of yet unknown prerequisites for the maintenance of both small and large circular DNA in cancers, but also in the context of other diseases and normal cellular development. |
Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome Wang, Min In: 2023. @article{noKey,
title = {Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome},
author = {Wang, Min},
url = {https://www.cell.com/cell/pdf/S0092-8674(23)00589-5.pdf},
doi = {https://doi.org/10.1016/j.cell.2023.05.037},
year = {2023},
date = {2023-01-01},
abstract = {The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype. |
Single-cell transcriptomics of a dynamic cell behavior in murine airways Kwok, Sheldon JJ In: 2023. @article{noKey,
title = {Single-cell transcriptomics of a dynamic cell behavior in murine airways},
author = {Kwok, Sheldon JJ},
url = {https://elifesciences.org/articles/76645},
doi = {https://doi.org/10.7554/eLife.76645},
year = {2023},
date = {2023-01-01},
abstract = {Despite advances in high-dimensional cellular analysis, the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge. We present a method that allows us to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells. This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Despite advances in high-dimensional cellular analysis, the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge. We present a method that allows us to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells. This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways. |
Interlaboratory Comparison Using Inactivated Authentic Sars-Cov-2 Variants as a Feasible Tool for Quality Control in Covid-19 Wastewater Monitoring Wilhelm, Alexander In: 2023. @article{noKey,
title = {Interlaboratory Comparison Using Inactivated Authentic Sars-Cov-2 Variants as a Feasible Tool for Quality Control in Covid-19 Wastewater Monitoring},
author = {Wilhelm, Alexander},
url = {https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4442612},
doi = {http://dx.doi.org/10.2139/ssrn.4442612},
year = {2023},
date = {2023-01-01},
abstract = {Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results.In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values.Despite the different procedures, a high concordance regarding the SARS-CoV-2 PCR quantification could be achieved with low variation between the workflows. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/mL wastewater and had limited predictive value when analyzing at a genome coverage below 60%.This interlaboratory test demonstrates that despite different extraction and analysis methods, a high agreement of the SARS-CoV-2 genome copy equivalents could be achieved. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results.In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values.Despite the different procedures, a high concordance regarding the SARS-CoV-2 PCR quantification could be achieved with low variation between the workflows. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/mL wastewater and had limited predictive value when analyzing at a genome coverage below 60%.This interlaboratory test demonstrates that despite different extraction and analysis methods, a high agreement of the SARS-CoV-2 genome copy equivalents could be achieved. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection. |
A framework for ultra-low input spatial tissue proteomics Makhmut, Anuar In: 2023. @article{noKey,
title = {A framework for ultra-low input spatial tissue proteomics},
author = {Makhmut, Anuar},
url = {https://www.biorxiv.org/content/10.1101/2023.05.13.540426v2.full},
doi = {https://doi.org/10.1101/2023.05.13.540426},
year = {2023},
date = {2023-01-01},
abstract = {Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultra-sensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultra-sensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research. |
Time-aligned hourglass gastrulation models in rabbit and mouse Mayshar, Yoav In: 2023. @article{noKey,
title = {Time-aligned hourglass gastrulation models in rabbit and mouse},
author = {Mayshar, Yoav},
url = {https://www.cell.com/cell/fulltext/S0092-8674(23)00471-3?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867423004713%3Fshowall%3Dtrue},
doi = {https://doi.org/10.1016/j.cell.2023.04.037},
year = {2023},
date = {2023-01-01},
abstract = {The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals. |
Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening Roy, Anu, Alhammad, Yousef M. In: 2022. @article{noKey,
title = {Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening},
author = {Roy, Anu, Alhammad, Yousef M.},
url = {https://www.sciencedirect.com/science/article/pii/S0166354222001139?casa_token=RshGcMd12jcAAAAA:WD7P4SRzuTcL4ga_b1yJNGRPlt64Xschlzswsdxh83HtjvC43ulfkWRQwojgQeiHjo0EjiZyBqg},
doi = {https://doi.org/10.1016/j.antiviral.2022.105344},
year = {2022},
date = {2022-01-01},
abstract = {The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen ∼38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100µM, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen ∼38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100µM, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors. |
Discovery and Mechanism of Small Molecule Inhibitors Selective for the Chromatin-Binding Domains of Oncogenic UHRF1 (Ed.) In: 2022. @article{noKey,
title = {Discovery and Mechanism of Small Molecule Inhibitors Selective for the Chromatin-Binding Domains of Oncogenic UHRF1},
url = {https://pubs.acs.org/doi/pdf/10.1021/acs.biochem.1c00698},
doi = {https://doi.org/10.1021/acs.biochem.1c00698},
year = {2022},
date = {2022-01-01},
abstract = {Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with histones in vitro and in cells. Together, our study provides insight into the critical role of the PHD finger in driving histone interactions, and demonstrates that targeting this domain through a specific binding pocket is a tractable strategy for UHRF1-histone inhibition.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with histones in vitro and in cells. Together, our study provides insight into the critical role of the PHD finger in driving histone interactions, and demonstrates that targeting this domain through a specific binding pocket is a tractable strategy for UHRF1-histone inhibition. |
