210 Citations
Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes but global profiling of protein complexes directly from human tissue samples has been limited In this study we utilized co-fractionation mass spectrometry CF-MS to map protein complexes within the post-mortem human brain with experimental replicates First we used concatenated anion and cation Ion Exchange Chromatography IEX to separate native protein complexes in fractions then proceeded with Data-Independent Acquisition DIA mass spectrometry to analyze the proteins in each fraction quantifying a total of proteins with overlapping in both replicates We improved DIA quantitative accuracy by ... More
Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes, but global profiling of protein complexes directly from human tissue samples has been limited. In this study, we utilized co-fractionation mass spectrometry (CF-MS) to map protein complexes within the post-mortem human brain with experimental replicates. First, we used concatenated anion and cation Ion Exchange Chromatography (IEX) to separate native protein complexes in 192 fractions, then proceeded with Data-Independent Acquisition (DIA) mass spectrometry to analyze the proteins in each fraction, quantifying a total of 4,804 proteins with 3,260 overlapping in both replicates. We improved DIA quantitative accuracy by implementing a constant amount of bovine serum albumin (BSA) in each fraction as an internal standard. Next, advanced computational pipelines, which integrate both a database-based complex analysis and an unbiased protein-protein interaction (PPI) search, were applied to identify protein complexes and construct protein-protein interaction networks in the human brain. Our study led to the identification of 486 protein complexes and 10,054 binary protein-protein interactions, which represents the first global profiling of human brain PPIs using CF-MS. Overall, this study offers a resource and tool for a wide range of human brain research, including the identification of disease-specific protein complexes in the future. Less
Cancer cells often exhibit DNA copy number aberrations and can vary widely in their ploidy Correct estimation of the ploidy of single-cell genomes is paramount for downstream analysis Based only on single-cell DNA sequencing information scAbsolute achieves accurate and unbiased measurement of single-cell ploidy and replication status including whole-genome duplications We demonstrate scAbsolute s capabilities using experimental cell multiplets a FUCCI cell cycle expression system and a benchmark against state-of-the-art methods scAbsolute provides a robust foundation for single-cell DNA sequencing analysis across different technologies and has the potential to enable improvements in a number of downstream analyses
Hematopoietic stem cells HSCs develop from the hemogenic endothelium HE in the aorta- gonads-and mesonephros AGM region and reside within Intra-aortic hematopoietic clusters IAHC along with hematopoietic progenitors HPC The signalling mechanisms that distinguish HSCs from HPCs are unknown Notch signaling is essential for arterial specification IAHC formation and HSC activity but current studies on how Notch segregates these different fates are inconsistent We now demonstrate that Notch activity is highest in a subset of GFI HSC-primed HE cells and is gradually lost with HSC maturation We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH ... More
Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta. Less
ADP-ribosyltransferases PARP and PARP play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations Recently a study showed that Histone PARylation Factor HPF forms a joint active site with PARP The interaction of HPF with PARP alters the modification site from Aspartate Glutamate to Serine which has been shown to be a key ADP-ribosylation event in the context of DNA damage Therefore disruption of PARP -HPF interaction could ... More
ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents. Less
Memory encodes past experiences thereby enabling future plans The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks These transcriptional signatures implicate neuropeptide and BDNF signalling MAPK and CREB activation ubiquitination pathways and synaptic connectivity as key components of long-term memory Notably upon long-term memory ... More
Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory. Less
In the fields of human health and agricultural research low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples However a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature In this study we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits the KAPA HyperPlus kit Roche the DNA Prep kit Illumina and an IDT kit using human DNA samples Metrics evaluated included imputation concordance with ... More
In the fields of human health and agricultural research, low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples. However, a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature. In this study, we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits—the KAPA HyperPlus kit (Roche), the DNA Prep kit (Illumina), and an IDT kit—using 96 human DNA samples. Metrics evaluated included imputation concordance with high-depth genotypes, coverage, duplication rates, time for library preparation, and additional optimization requirements. Despite slightly elevated duplication rates in IDT kits, we find that all four kits perform well in terms of imputation accuracy, with IDT kits being only marginally less performant than Illumina and Roche kits. Laboratory handling of the kits was similar: thus, the choice of a kit will largely depend on (1) existing or planned infrastructure, such as liquid handling capabilities, (2) whether a specific characteristic is desired, such as the use of full-length adapters, shorter processing times, or (3) use case, for instance, long vs short read sequencing. Our findings offer a comprehensive resource for both commercial and research workflows of low-cost library preparation methods suitable for high-throughput low coverage whole genome sequencing. Less
Exposure to environmental pollutants is linked to numerous toxic outcomes warranting concern about the effect of pollutants on human health To assess the threat of pollutant exposure it is essential to understand their biological activity Unfortunately gaps remain for many pollutants specific biological activity and molecular targets A superfamily of signaling proteins G-protein-coupled receptors GPCRs has been shown as potential targets for pollutant activity However research investigating the pollutant activity at the GPCRome is scarce This work explores pollutant activity across a library of human GPCRs by leveraging modern high-throughput screening techniques devised for drug discovery and pharmacology We designed ... More
Exposure to environmental pollutants is linked to numerous toxic outcomes, warranting concern about the effect of pollutants on human health. To assess the threat of pollutant exposure, it is essential to understand their biological activity. Unfortunately, gaps remain for many pollutants’ specific biological activity and molecular targets. A superfamily of signaling proteins, G-protein-coupled receptors (GPCRs), has been shown as potential targets for pollutant activity. However, research investigating the pollutant activity at the GPCRome is scarce. This work explores pollutant activity across a library of human GPCRs by leveraging modern high-throughput screening techniques devised for drug discovery and pharmacology. We designed and implemented a pilot screen of eight pollutants at 314 human GPCRs and discovered specific polychlorinated biphenyl (PCB) activity at sphingosine-1-phosphate and melatonin receptors. The method utilizes open-source resources available to academic and governmental institutions to enable future campaigns that screen large numbers of pollutants. Thus, we present a novel high-throughput approach to assess the biological activity and specific targets of pollutants. Less
We herein describe the development and application of a modular technology platform which incorporates recent advances in plate-based microscale chemistry automated purification in situ quantification and robotic liquid handling to enable rapid access to high-quality chemical matter already formatted for assays In using microscale chemistry and thus consuming minimal chemical matter the platform is not only efficient but also follows green chemistry principles By reorienting existing high-throughput assay technology the platform can generate a full package of relevant data on each set of compounds in every learning cycle The multiparameter exploration of chemical and property space is hereby driven by ... More
We herein describe the development and application of a modular technology platform which incorporates recent advances in plate-based microscale chemistry, automated purification, in situ quantification, and robotic liquid handling to enable rapid access to high-quality chemical matter already formatted for assays. In using microscale chemistry and thus consuming minimal chemical matter, the platform is not only efficient but also follows green chemistry principles. By reorienting existing high-throughput assay technology, the platform can generate a full package of relevant data on each set of compounds in every learning cycle. The multiparameter exploration of chemical and property space is hereby driven by active learning models. The enhanced compound optimization process is generating knowledge for drug discovery projects in a time frame never before possible. Less
Predictive drug testing of patient-derived tumor organoids PDTOs holds promise for personalizing treatment of metastatic colorectal cancer mCRC but prospective data are limited to chemotherapy regimens with conflicting results We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options In an Australian community cohort PDTO predictions based on treatment-naive patients n and response rates from first-line mCRC clinical trials achieve accuracy for forecasting responses in patients receiving palliative treatments patients treatments Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment AGITG FORECAST- n patients Resistant predictions ... More
Predictive drug testing of patient-derived tumor organoids (PDTOs) holds promise for personalizing treatment of metastatic colorectal cancer (mCRC), but prospective data are limited to chemotherapy regimens with conflicting results. We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options. In an Australian community cohort, PDTO predictions based on treatment-naive patients (n = 56) and response rates from first-line mCRC clinical trials achieve 83% accuracy for forecasting responses in patients receiving palliative treatments (18 patients, 29 treatments). Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment, AGITG FORECAST-1 (n = 30 patients). “Resistant” predictions are associated with inferior progression-free survival; misclassification rates are similar by regimen. Liver metastases are the optimal site for sampling, with testing achievable within 7 weeks for 68.8% cases. Our findings indicate that PDTO drug panel testing can provide predictive information for multifarious standard-of-care therapies for mCRC. Less
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection It is responsible for US deaths annually 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 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 ... More
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. Less
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. Less
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 ... More
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. Less
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 quantified proteins from single hepatocyte contours and nearly proteins from -cell ... More
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. Less
The COVID- pandemic caused by severe acute respiratory syndrome coronavirus SARS-CoV- virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV- variants or novel CoVs Here we describe small molecule inhibitors for SARS-CoV- Mac which counters ADP-ribosylation mediated innate immune responses The compounds inhibiting Mac were discovered through high-throughput screening HTS using a protein FRET-based competition assay and the best hit compound had an IC of M Three validated HTS hits have the same -amide- -methylester thiophene scaffold and the scaffold was selected for structure-activity relationship SAR studies through commercial and synthesized ... More
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. Less
Wastewater-based SARS-CoV- 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 ... More
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. Less
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 ... More
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. Less
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 ... More
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. Less
Receptor-interacting protein kinases and RIPK and RIPK 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 and 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 RIPK and RIPK in complex with known quinazoline or quinoline inhibitors Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs RIPK - shed ... More
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. Less
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 Rev translesion polymerase activity Inactivating Rev did not alter the nAt trinucleotide preference suggesting it may be an intrinsic specificity of the chemical reaction ... More
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. Less
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 -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 -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 -fold in the Cs but ... More
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. Less
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 ... More
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. Less
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 and and compared the species using a framework for time-resolved single-cell differentiation-flows analysis We find convergence toward similar cell-state compositions at E supported by the quantitatively conserved expression of transcription factors despite divergence in surrounding trophoblast and hypoblast signaling However we ... More
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. Less
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
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 vs kg N m d lead to different combinations of anammox bacteria and anaerobic methanotrophs Candidatus Methanoperedens and Candidatus Methylomirabilis which play primary roles for ... More
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. Less
Described herein are methods for stratifying and evaluating melanoma treatment response in a subject using single-cell RNA sequencing scRNA-seq and a two-step deconvolution analysis and optionally administering a treatment depending on the results Embodiment described herein are methods for stratifying and evaluating melanoma treatment response in a subject based on single cell or bulk RNA sequencing bulk transcriptome profiling and or transcript counting and a two-step deconvolution analysis and optionally administering a treatment depending on the results
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 Methanocorpusculum ... More
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. Less
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. Less
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 bacterial and archaeal species strains across days Results Due to the remarkable increase decrease of constituent microbial population densities ... More
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. Less
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. Less
A rare coding variant in PLC P R 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 PLC has been suggested as a potential therapeutic target for the prevention and treatment of LOAD Additionally PLC has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLC activity have been identified Here pharmacological inhibition may provide a therapeutic effect In order to facilitate our ... More
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. Less
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 CD THY lo cells were enriched for radial glia which robustly engrafted and differentiated into all three neural lineages in the mouse brain THY hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial ... More
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. Less
Ubiquitination is a complex and reversible protein post-translational modification in which the subsequent action of enzymes belonging to three different families broadly referred to as E E and E results in the covalent linking of ubiquitin to a target protein While this linkage is canonically an isopeptide bond between the C-terminus of ubiquitin and the lysine residue of the target protein Ser Thr and Tyr can also be susceptible to ubiquitination through an oxyester bond Once ubiquitinated multiple units of ubiquitin can be attached to the initial ubiquitin thus extending it to a chain of ubiquitins Ubiquitination regulates multiple cellular ... More
Ubiquitination is a complex and reversible protein post-translational modification in which the subsequent action of enzymes belonging to three different families, broadly referred to as E1, E2 and E3, results in the covalent linking of ubiquitin to a target protein. While this linkage is canonically an isopeptide bond between the C-terminus of ubiquitin and the lysine residue of the target protein, Ser, Thr, and Tyr can also be susceptible to ubiquitination through an oxyester bond. Once ubiquitinated, multiple units of ubiquitin can be attached to the initial ubiquitin thus extending it to a chain of ubiquitins. Ubiquitination regulates multiple cellular processes, but it is best known as a modification that targets proteins for proteasomal degradation following the formation poly-ubiquitin chains linked through lysine 48 or 63 of ubiquitin. Dysregulation of ubiquitination has been associated with multiple types of cancer and efforts have been carried out to develop technologies that lead to the identification of inhibitors of the enzymes involved in the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor auto-ubiquitination of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a robust signal window with a robust average Z’ factor of 0.76. From a validatory screening experiment we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. Additionally, we have expanded the system to study deubiquitinases such as USP28 that lead to reduction of FRET due to hydrolysis of fluorescent poly-Ub chains. Less
Analyzing proteins from single cells by tandem mass spectrometry MS has recently become technically feasible While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design sample preparation data acquisition and data analysis We expect that broadly accepted community guidelines and standardized metrics will enhance rigor data quality and alignment between laboratories Here we propose best practices quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics
Pure bacterial cultures remain essential for detailed experimental and mechanistic studies in microbiome research and traditional methods to isolate individual bacteria from complex microbial ecosystems are labor-intensive difficult-to-scale and lack phenotype genotype integration Here we describe an open-source high-throughput robotic strain isolation platform for the rapid generation of isolates on demand We develop a machine learning approach that leverages colony morphology and genomic data to maximize the diversity of microbes isolated and enable targeted picking of specific genera Application of this platform on fecal samples from humans yields personalized gut microbiome biobanks totaling isolates that represented of all abundant taxa ... More
Pure bacterial cultures remain essential for detailed experimental and mechanistic studies in microbiome research, and traditional methods to isolate individual bacteria from complex microbial ecosystems are labor-intensive, difficult-to-scale and lack phenotype–genotype integration. Here we describe an open-source high-throughput robotic strain isolation platform for the rapid generation of isolates on demand. We develop a machine learning approach that leverages colony morphology and genomic data to maximize the diversity of microbes isolated and enable targeted picking of specific genera. Application of this platform on fecal samples from 20 humans yields personalized gut microbiome biobanks totaling 26,997 isolates that represented >80% of all abundant taxa. Spatial analysis on >100,000 visually captured colonies reveals cogrowth patterns between Ruminococcaceae, Bacteroidaceae, Coriobacteriaceae and Bifidobacteriaceae families that suggest important microbial interactions. Comparative analysis of 1,197 high-quality genomes from these biobanks shows interesting intra- and interpersonal strain evolution, selection and horizontal gene transfer. This culturomics framework should empower new research efforts to systematize the collection and quantitative analysis of imaging-based phenotypes with high-resolution genomics data for many emerging microbiome studies. Less
Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication DNA repair and telomere maintenance Due to their essential role they are potential targets for novel anticancer and antimicrobial drugs but have so far been little exploited Here we present a simple and versatile real-time exonuclease assay based on -aminopurine an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both and DNA ends over a wide range of protein activities and suitable for a high-throughput inhibitor ... More
Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication, DNA repair, and telomere maintenance. Due to their essential role, they are potential targets for novel anticancer and antimicrobial drugs but have so far been little exploited. Here, we present a simple and versatile real-time exonuclease assay based on 2-aminopurine, an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA. We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both 3′ and 5′ DNA ends over a wide range of protein activities and suitable for a high-throughput inhibitor screening campaign. Using our assay, we discover a novel inhibitor of the Mycobacterium tuberculosis PHP-exonuclease that is part of the replicative DNA polymerase DnaE1. Hence, our novel assay will be a useful tool for high-throughput screening for novel exonuclease inhibitors that may interfere with DNA replication or DNA maintenance. Less
Breastmilk is thought to influence the infant gut by supplying prebiotics in the form of human milk oligosaccharides and potentially seeding the gut with breastmilk microbes However the presence of a breastmilk microbiota and origins of these microbes are still debated As a pilot study we assessed the microbes present in expressed breastmilk at six-weeks postpartum using shotgun metagenomic sequencing in a heterogenous cohort of women who delivered by vaginal n and caesarean delivery n In addition we estimated the microbial load of breastmilk at six-weeks post-partum with quantitative PCR targeting the S rRNA gene Breastmilk at six-weeks postpartum had ... More
Breastmilk is thought to influence the infant gut by supplying prebiotics in the form of human milk oligosaccharides and potentially seeding the gut with breastmilk microbes. However, the presence of a breastmilk microbiota and origins of these microbes are still debated. As a pilot study, we assessed the microbes present in expressed breastmilk at six-weeks postpartum using shotgun metagenomic sequencing in a heterogenous cohort of women who delivered by vaginal (n = 8) and caesarean delivery (n = 8). In addition, we estimated the microbial load of breastmilk at six-weeks post-partum with quantitative PCR targeting the 16S rRNA gene. Breastmilk at six-weeks postpartum had a low microbial mass, comparable with PCR no-template and extraction controls. Microbes identified through metagenomic sequencing were largely consistent with skin and oral microbes, with four samples returning no identifiable bacterial sequences. Our results do not provide convincing evidence for the existence of a breastmilk microbiota at six-weeks postpartum. It is more likely that microbes present in breastmilk are sourced by ejection from the infant’s mouth and from surrounding skin, as well as contamination during sampling and processing. Less
This high-throughput protocol details the steps to extract protein from Gram-negative bacteria Gram-positive bacteria or non-filamentous fungi in -well plate format for quantitative proteomic workflows This protocol uses a bench-top automated liquid dispenser but the volumes and times also apply to manual and multi-channel pipetter use This protocol is designed for lab-based culture conditions and synthetic community experiments where complex sample matrices are minimized Additional sample preservation and or protein extraction methods may be required for environmental samples e g feces soil to minimize protein degradation and maintain sample integrity
Background Xylem the most abundant tissue on Earth is responsible for lateral growth in plants Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells In most angiosperms fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support while both functions are performed by tracheids in other vascular plants such as gymnosperms Little is known about the developmental programs and evolutionary relationships of these xylem cell types Results Through both single-cell and laser capture microdissection transcriptomic profiling we determine the developmental lineages of ray and fusiform cells in ... More
Background
Xylem, the most abundant tissue on Earth, is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells. In most angiosperms, fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed by tracheids in other vascular plants such as gymnosperms. Little is known about the developmental programs and evolutionary relationships of these xylem cell types.
Results
Through both single-cell and laser capture microdissection transcriptomic profiling, we determine the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Based on cross-species analyses of single-cell clusters and overlapping trajectories, we reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages, whereas the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed trait, exhibit strong transcriptomic similarity to vessel elements rather than libriform fibers.
Conclusions
This evo-devo framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history. Less
Xylem, the most abundant tissue on Earth, is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells. In most angiosperms, fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed by tracheids in other vascular plants such as gymnosperms. Little is known about the developmental programs and evolutionary relationships of these xylem cell types.
Results
Through both single-cell and laser capture microdissection transcriptomic profiling, we determine the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Based on cross-species analyses of single-cell clusters and overlapping trajectories, we reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages, whereas the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed trait, exhibit strong transcriptomic similarity to vessel elements rather than libriform fibers.
Conclusions
This evo-devo framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history. Less
Single-cell multi-omics can provide a unique perspective on tumor cellular heterogeneity Most previous single-cell whole-genome RNA sequencing scWGS-RNA-seq methods demonstrate utility with intact cells from fresh samples Among them many are not applicable to frozen samples that cannot produce intact single-cell suspensions We have developed scONE-seq a versatile scWGS-RNA-seq method that amplifies single-cell DNA and RNA without separating them from each other and hence is compatible with frozen biobanked samples We benchmarked scONE-seq against existing methods using fresh and frozen samples to demonstrate its performance in various aspects We identified a unique transcriptionally normal-like tumor clone by analyzing a -year ... More
Single-cell multi-omics can provide a unique perspective on tumor cellular heterogeneity. Most previous single-cell whole-genome RNA sequencing (scWGS-RNA-seq) methods demonstrate utility with intact cells from fresh samples. Among them, many are not applicable to frozen samples that cannot produce intact single-cell suspensions. We have developed scONE-seq, a versatile scWGS-RNA-seq method that amplifies single-cell DNA and RNA without separating them from each other and hence is compatible with frozen biobanked samples. We benchmarked scONE-seq against existing methods using fresh and frozen samples to demonstrate its performance in various aspects. We identified a unique transcriptionally normal-like tumor clone by analyzing a 2-year frozen astrocytoma sample, demonstrating that performing single-cell multi-omics interrogation on biobanked tissue by scONE-seq could enable previously unidentified discoveries in tumor biology. Less
Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single nucleotide polymorphisms SNPs The main limitation of current methods is the cost is too high to screen breeding populations Molecular inversion probes MIPs is a targeted genotyping-by-sequencing method that could be used for soybeans that is both cost effective high-throughput and provides high data quality to screen breeder s germplasm for genomic selection A K MIP SNP set was developed for soybean with uniformly distributed markers across the genome The SNPs were selected to maximize the number ... More
Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single nucleotide polymorphisms (SNPs). The main limitation of current methods is the cost is too high to screen breeding populations. Molecular inversion probes (MIPs) is a targeted genotyping-by-sequencing method that could be used for soybeans that is both cost effective, high-throughput, and provides high data quality to screen breeder’s germplasm for genomic selection. A 1K MIP SNP set was developed for soybean with uniformly distributed markers across the genome. The SNPs were selected to maximize the number of informative markers in germplasm being tested in soybean breeding programs located in the North Central and Mid-South regions of the United States. The 1K SNP MIP set was tested on diverse germplasm and a recombinant inbred line population. Targeted sequencing with MIPs obtained an 85% enrichment for the targeted SNPs. MIP’s genotyping accuracy was 93% overall while homozoygous call accuracy was 98% with less than 10% missing data. The accuracy of MIPs combined with its low per sample cost makes it a powerful tool to enable genomic selection within soybean breeding programs. Less
Microfluidic paper-based analytical devices microPADs are an emerging platform for point-of-care assays with potential applications ranging from medical diagnostics to environmental monitoring A standard approach for signal generation and amplification on microPADs is the use of enzymes and chromogenic substrates which produce colored products and enable the qualitative and quantitative detection of analytes Horseradish peroxidase HRP is one of the most commonly used enzymes for this application While a wide variety of chromogenic substrates and substrate solutions are available for HRP these were all originally developed and optimized for other platforms and have not been systematically compared on microPADs In ... More
Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care assays with potential applications ranging from medical diagnostics to environmental monitoring. A standard approach for signal generation and amplification on microPADs is the use of enzymes and chromogenic substrates, which produce colored products and enable the qualitative and quantitative detection of analytes. Horseradish peroxidase (HRP) is one of the most commonly used enzymes for this application. While a wide variety of chromogenic substrates and substrate solutions are available for HRP, these were all originally developed and optimized for other platforms and have not been systematically compared on microPADs. In this work, 33 chromogenic substrate solutions for HRP containing one of six different substrate molecules were evaluated in a standardized colorimetric assay and compared on the basis of limit of detection (LOD), lower limit of quantitation (LLOQ), upper limit of quantitation (ULOQ), sensitivity, dynamic range, stability, and cost. While the substrates tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) were found to achieve the best overall analytical performance, each substrate had characteristics that may make them appealing for specific applications. The results of this work will facilitate the selection of substrate molecules and the formulation of substrate solutions for future colorimetric paper-based assays involving HRP. Less
Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets Despite recent advances in the field efforts to discover such compounds have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different PARP enzymes of the ARTD family We here expanded the methods and engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold Compared to the free enzymes the scaffold-based system results in an improved activity for the tested PARPs due to improved ... More
Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover such compounds have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different PARP enzymes of the ARTD family. We here expanded the methods and engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their enhanced activity using a simple and easily accessible homogeneous NAD+ conversion assay, facilitating its automation to reduce the assay volume and lowering the assay costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity and it can be applied to compound screening campaigns as well as inhibitor profiling. Less
Single-cell nucleosome methylome and transcriptome scNMT sequencing is a recently developed method that allows multiomics profiling of single cells In this scNMT protocol we describe profiling of cells from mouse brain and pancreatic organoids using liquid handling platforms to increase throughput from -well to -well plate format Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq protocol to obtain higher numbers of detected genes and genomic DNA gDNA CpGs per cell We outline normalization steps to optimally distribute per-cell sequencing depth
The recent discovery of comammox complete ammonia oxidation Nitrospira has upended the long-held nitrification paradigm Although comammox Nitrospira have been identified in wastewater treatment systems the conditions for their dominance over canonical ammonia oxidizers remain unclear Here we report the dominance of comammox Nitrospira in a moving bed biofilm reactor MBBR fed with synthetic mainstream wastewater Integrated S rRNA gene amplicon sequencing fluorescence in situ hybridization FISH and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen DO concentration above mg O L The dominance of comammox Nitrospira over canonical ... More
The recent discovery of comammox (complete ammonia oxidation) Nitrospira has upended the long-held nitrification paradigm. Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02–0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospira were identified, which are phylogenetically close to Candidatus Nitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR. Less
We herein report the development of an automation platform for rapid purification and quantification of chemical libraries including reformatting of chemical matter to mM DMSO stock solutions This fully integrated workflow features tailored conditions for preparative reversed-phase RP HPLC-MS on microscale based on analytical data online fraction QC and CAD-based quantification as well as automated reformatting to enable rapid purification of chemical libraries This automated workflow is entirely solution-based eliminating the need to weigh or handle solids This increases process efficiency and creates a link between high-throughput synthesis and profiling of novel chemical matter with respect to biological and physicochemical ... More
We herein report the development of an automation platform for rapid purification and quantification of chemical libraries including reformatting of chemical matter to 10 mM DMSO stock solutions. This fully integrated workflow features tailored conditions for preparative reversed-phase (RP) HPLC-MS on microscale based on analytical data, online fraction QC and CAD-based quantification as well as automated reformatting to enable rapid purification of chemical libraries. This automated workflow is entirely solution-based, eliminating the need to weigh or handle solids. This increases process efficiency and creates a link between high-throughput synthesis and profiling of novel chemical matter with respect to biological and physicochemical properties in relevant assays. Less
Polyurethane-based hydrogels are relatively inexpensive and mechanically robust biomaterials with ideal properties for various applications including drug delivery prosthetics implant coatings soft robotics and tissue engineering In this report we present a simple method for synthesizing and casting biocompatible polyurethane-poly ethylene glycol PU-PEG hydrogels with tunable mechanical properties non-fouling characteristics and sustained tolerability as an implantable material or coating The hydrogels are synthesized via a simple one-pot method using commercially available precursors and low toxicity solvents and reagents yielding a consistent and biocompatible gel platform primed for long-term biomaterial applications The mechanical and physical properties of the gels are easily ... More
Polyurethane-based hydrogels are relatively inexpensive and mechanically robust biomaterials with ideal properties for various applications, including drug delivery, prosthetics, implant coatings, soft robotics, and tissue engineering. In this report, we present a simple method for synthesizing and casting biocompatible polyurethane-poly(ethylene glycol) (PU-PEG) hydrogels with tunable mechanical properties, non-fouling characteristics, and sustained tolerability as an implantable material or coating. The hydrogels are synthesized via a simple one-pot method using commercially available precursors and low toxicity solvents and reagents, yielding a consistent and biocompatible gel platform primed for long-term biomaterial applications. The mechanical and physical properties of the gels are easily controlled by varying the curing concentration, producing networks with complex shear moduli of 0.82 kPa – 190 kPa, similar to a range of human soft tissues. When evaluated against a mechanically-matched PDMS formulation the PU-PEG hydrogels demonstrated favorable non-fouling characteristics including comparable adsorption of plasma proteins (albumin and fibrinogen) and significantly reduced cellular adhesion. Moreover, preliminary murine implant studies reveal a mild foreign body response after 41 days. Due to the tunable mechanical properties, excellent biocompatibility, and sustained in vivo tolerability of these hydrogels, we propose that this method offers a simplified platform for fabricating soft PU-based biomaterials for a variety of applications. Less
Mice deficient for all ten-eleven translocation TET genes exhibit early gastrulation lethality However separating cause and effect in such embryonic failure is challenging To isolate cell-autonomous effects of TET loss we used temporal single-cell atlases from embryos with partial or complete mutant contributions Strikingly when developing within a wild-type embryo Tet-mutant cells retain near-complete differentiation potential whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential We map de-repressions of early epiblast factors e g Dppa and Gdf and failure to activate multiple signaling from nascent mesoderm Lefty FGF and Notch as likely ... More
Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications. Less
A series of amino acid based H-pyrrolo d pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV- nsp macrodomain Mac an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis Structure activity studies identified compound c as a low-micromolar inhibitor of Mac in two ADP-ribose binding assays This compound also demonstrated inhibition in an enzymatic assay of Mac and displayed a thermal shift comparable to ADPr in the melting temperature of Mac supporting binding to the target protein A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network ... More
A series of amino acid based 7H-pyrrolo[2,3–d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization. Less
The SARS-CoV- infection cycle is a multistage process that relies on functional interactions between the host and the pathogen Here we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA '-O-ribose cap needed for viral immune escape We find that the host cap '-O-ribose methyltransferase MTr can compensate for loss of viral NSP methyltransferase in facilitating virus replication Concomitant inhibition of MTr and NSP efficiently suppresses SARS-CoV- replication Using in silico target-based drug screening we identify a bispecific MTr NSP inhibitor with anti-SARS-CoV- activity in vitro and in vivo but with unfavorable ... More
The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19. Less
Buffalo flies Haematobia irritans exigua are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas most commonly on the medial canthus of the eye along the lateral and ventral neck and on the abdomen of cattle For closely related horn flies Haematobia irritans irritans Staphylococcus aureus has been suggested as a contributing factor in the development of lesions To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions swabs were taken from ... More
Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions. Less
Chemical proteomics studies the effects of drugs upon a cellular proteome Due to the complexity and diversity of tumors the response of cancer cells to drugs is also heterogeneous and thus proteome analysis at the single-cell level is needed Here we demonstrate that single-cell proteomics techniques have become quantitative enough to tackle the drug effects on target proteins enabling single-cell chemical proteomics SCCP Using SCCP we studied here the time-resolved response of individual adenocarcinoma A cells to anticancer drugs methotrexate camptothecin and tomudex revealing the early emergence of cellular subpopulations committed and uncommitted to death As a novel and useful ... More
Chemical proteomics studies the effects of drugs upon a cellular proteome. Due to the complexity and diversity of tumors, the response of cancer cells to drugs is also heterogeneous, and thus, proteome analysis at the single-cell level is needed. Here, we demonstrate that single-cell proteomics techniques have become quantitative enough to tackle the drug effects on target proteins, enabling single-cell chemical proteomics (SCCP). Using SCCP, we studied here the time-resolved response of individual adenocarcinoma A549 cells to anticancer drugs methotrexate, camptothecin, and tomudex, revealing the early emergence of cellular subpopulations committed and uncommitted to death. As a novel and useful approach to exploring the heterogeneous response to drugs of cancer cells, SCCP may prove to be a breakthrough application for single-cell proteomics. Less
Introduction Advancements in technology and communication have revolutionised the st century with the introduction of mobile phones and smartphones These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination Aim To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff Methods Twenty-six mobile phones of health care staff were swabbed DNA extraction for downstream next generation sequencing shotgun ... More
Introduction. Advancements in technology and communication have revolutionised the 21st century with the introduction of mobile phones and smartphones. These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour. Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination. Aim. To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff. Methods. Twenty-six mobile phones of health care staff were swabbed. DNA extraction for downstream next generation sequencing shotgun metagenomic microbial profiling was performed. Survey questionnaires were handed to the staff to collect information on mobile phone usage and users’ behaviours. Results. A total of 11259 organisms derived from 26 phones were found with 2096 genes coding for antibiotic resistance and virulent factors. These organisms corresponded to 5717 bacteria, 675 fungi, 93 protists, 320 viruses, 4456 bacteriophages. The survey of medical staff showed that 46% (12/26) of the participants used their mobile phones in the bathroom. Discussion/conclusion. Mobile phones are vectors of microbes and can contribute to microbial dissemination and nosocomial diseases worldwide. As fomites, mobile phones that are not decontaminated may pose serious risks for public health and biosecurity. Less
Current single-cell RNA sequencing scRNA-seq methods with high cellular throughputs sacrifice full-transcript coverage and often sensitivity Here we describe Smart-seq xpress which miniaturizes and streamlines the Smart-seq protocol to substantially reduce reagent use and increase cellular throughput Smart-seq xpress analysis of peripheral blood mononuclear cells resulted in a granular atlas complete with common and rare cell types Compared with droplet-based single-cell RNA sequencing that sequences RNA ends the additional full-transcript coverage revealed cell-type-associated isoform variation