Publications

@article{noKey,

title = {A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes},

author = {Visvanathan, Ramya},

url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0299541},

doi = {https://doi.org/10.1371/journal.pone.0299541},

year  = {2024},

date = {2024-01-01},

abstract = {The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {scAbsolute: measuring single-cell ploidy and replication status},

author = {Schneider, Michael P.},

url = {https://link.springer.com/article/10.1186/s13059-024-03204-y},

doi = {https://doi.org/10.1186/s13059-024-03204-y},

year  = {2024},

date = {2024-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate},

author = {Thambyrajah, Roshana},

url = {https://www.nature.com/articles/s41467-024-45716-y},

doi = {https://doi.org/10.1101/2023.04.19.537430},

year  = {2024},

date = {2024-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {High-throughput screening assay for PARP-HPF1 interaction inhibitors to affect DNA damage repair},

author = {Dhakar, Saurabh S.},

url = {https://www.nature.com/articles/s41598-024-54123-8},

doi = {https://doi.org/10.1038/s41598-024-54123-8},

year  = {2024},

date = {2024-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Transcriptomic Signatures of WNT-Driven Pathways and Granulosa Cell-Oocyte Interactions during Primordial Follicle Activation},

author = {Takase, Hinako M.},

url = {https://www.biorxiv.org/content/10.1101/2024.02.08.579446v2},

doi = {https://doi.org/10.1101/2024.02.08.579446},

year  = {2024},

date = {2024-01-01},

abstract = {Primordial follicle activation (PFA) is a pivotal event in female reproductive biology, coordinating the transition from quiescent to growing follicles. This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells (GCs) and oocytes with the progression of the PFA process. Wntless (Wls) conditional knockout (cKO) mice served as a unique model, suppressing the transition from pre-GCs to GCs, and disrupting somatic cell-derived WNT signaling in the ovary. Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice, leading to a divergent trajectory, while oocytes exhibited modest transcriptomic alterations. Subpopulation analysis identified the molecular pathways affected by WNT signaling on GC maturation, along with specific gene signatures linked to dormant and activated oocytes. Despite minimal evidence of continuous up-regulation of dormancy-related genes in oocytes, the loss of WNT signaling in (pre-)GCs impacted gene expression in oocytes even before PFA, subsequently influencing them globally. The infertility observed in Wls cKO mice was attributed to compromised GC-oocyte molecular crosstalk and the microenvironment for oocytes. Our study highlights the pivotal role of the WNT-signaling pathway and its molecular signature, emphasizing the importance of intercellular crosstalk between (pre-)GCs and oocytes in orchestrating folliculogenesis.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Spatial transcriptomics reveal neuron–astrocyte synergy in long-term memory},

author = {Sun, Wenfei},

url = {https://www.nature.com/articles/s41586-023-07011-6},

doi = {https://doi.org/10.1038/s41586-023-07011-6},

year  = {2024},

date = {2024-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {A comparison between low-cost library preparation kits for low coverage sequencing},

author = {Stewart et, Caitlin M.},

url = {https://www.biorxiv.org/content/10.1101/2024.01.30.578044v1},

doi = {https://doi.org/10.1101/2024.01.30.578044},

year  = {2024},

date = {2024-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Global genomic diversity of Pseudomonas aeruginosa in bronchiectasis},

author = {Harrington, Niamh E.},

url = {https://www.biorxiv.org/content/10.1101/2024.01.30.577916v1},

doi = {https://doi.org/10.1101/2024.01.30.577916},

year  = {2024},

date = {2024-01-01},

abstract = {Background Pseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis (CF) and bronchiectasis, associated with poorer outcomes. Unlike CF, bronchiectasis has been a neglected disease. More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P. aeruginosa infections to guide novel and improved treatments.



Methods We have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections.



Results We observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms.



Conclusions Our findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes.



Summary The largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {MicroCycle: An Integrated and Automated Platform to Accelerate Drug Discovery},

author = {Brocklehurst, Cara E.},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.3c02029},

doi = {https://doi.org/10.1021/acs.jmedchem.3c02029},

year  = {2024},

date = {2024-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Prevotella are major contributors of sialidases in the human vaginal microbiome},

author = {Pelayo, Paula},

url = {https://www.biorxiv.org/content/10.1101/2024.01.09.574895v1},

doi = {https://doi.org/10.1101/2024.01.09.574895},

year  = {2024},

date = {2024-01-01},

abstract = {Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis. These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment. Prior biochemical studies of vaginal bacterial sialidases have focused solely on the bacterial vaginosis-associated organism Gardnerella vaginalis. Despite their implications for sexual and reproductive health, sialidases from other vaginal bacteria have not been characterized. Here, we show that vaginal Prevotella species produce active sialidases that possess variable activity toward mucin. These sialidases are highly conserved across clades of Prevotella from different geographies, hinting at their importance globally. Finally, we find that Prevotella sialidases, including mucin-degrading enzymes from Prevotella timonensis, are highly prevalent and abundant in human vaginal metagenomes and metatranscriptomes, Together, our results identify Prevotella as a critical source of sialidases in the vaginal microbiome, improving our understanding of this detrimental bacterial activity.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Defining the function of disease variants with CRISPR editing and multimodal single cell sequencing},

author = {Baglaenko, Yuriy},

url = {https://www.biorxiv.org/content/10.1101/2024.03.28.587175v1.abstract},

doi = {https://doi.org/10.1101/2024.03.28.587175},

year  = {2024},

date = {2024-01-01},

abstract = {Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Isolation and characterisation of novel Methanocorpusculum species indicates the genus is ancestrally host-associated},

author = {Volmer, James G.},

url = {https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-023-01524-2},

doi = {https://doi.org/10.1186/s12915-023-01524-2},

year  = {2023},

date = {2023-01-01},

abstract = {Background

With an increasing interest in the manipulation of methane produced from livestock cultivation, the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with ‘low-methane’ emitters. Previously, marsupial species were shown to be enriched for novel lineages of Methanocorpusculum, as well as Methanobrevibacter, Methanosphaera, and Methanomassiliicoccales. Despite sporadic reports of Methanocorpusculum from stool samples of various animal species, there remains little information on the impacts of these methanogens on their hosts.



Results

Here, we characterise novel host-associated species of Methanocorpusculum, to explore unique host-specific genetic factors and their associated metabolic potential. We performed comparative analyses on 176 Methanocorpusculum genomes comprising 130 metagenome-assembled genomes (MAGs) recovered from 20 public animal metagenome datasets and 35 other publicly available Methanocorpusculum MAGs and isolate genomes of host-associated and environmental origin. Nine MAGs were also produced from faecal metagenomes of the common wombat (Vombatus ursinus) and mahogany glider (Petaurus gracilis), along with the cultivation of one axenic isolate from each respective animal; M. vombati (sp. nov.) and M. petauri (sp. nov.).



Conclusions

Through our analyses, we substantially expand the available genetic information for this genus by describing the phenotypic and genetic characteristics of 23 host-associated species of Methanocorpusculum. These lineages display differential enrichment of genes associated with methanogenesis, amino acid biosynthesis, transport system proteins, phosphonate metabolism, and carbohydrate-active enzymes. These results provide insights into the differential genetic and functional adaptations of these novel host-associated species of Methanocorpusculum and suggest that this genus is ancestrally host-associated.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Assessment of melanoma therapy response},

author = {L, Robert},

url = {https://patents.google.com/patent/US20230112964A1/en},

doi = {US20230112964A1},

year  = {2023},

date = {2023-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Core species and interactions prominent in fish-associated microbiome dynamics},

author = {Yajima, Daii},

url = {https://link.springer.com/article/10.1186/s40168-023-01498-x},

doi = {https://doi.org/10.1186/s40168-023-01498-x},

year  = {2023},

date = {2023-01-01},

abstract = {Background

In aquatic ecosystems, the health and performance of fish depend greatly on the dynamics of microbial community structure in the background environment. Nonetheless, finding microbes with profound impacts on fish’s performance out of thousands of candidate species remains a major challenge.



Methods

We examined whether time-series analyses of microbial population dynamics could illuminate core components and structure of fish-associated microbiomes in the background (environmental) water. By targeting eel-aquaculture-tank microbiomes as model systems, we reconstructed the population dynamics of the 9605 bacterial and 303 archaeal species/strains across 128 days.



Results

Due to the remarkable increase/decrease of constituent microbial population densities, the taxonomic compositions of the microbiome changed drastically through time. We then found that some specific microbial taxa showed a positive relationship with eels’ activity levels even after excluding confounding effects of environmental parameters (pH and dissolved oxygen level) on population dynamics. In particular, a vitamin-B12-producing bacteria, Cetobacterium somerae, consistently showed strong positive associations with eels’ activity levels across the replicate time series of the five aquaculture tanks analyzed. Network theoretical and metabolic modeling analyses further suggested that the highlighted bacterium and some other closely-associated bacteria formed “core microbiomes” with potentially positive impacts on eels.



Conclusions

Overall, these results suggest that the integration of microbiology, ecological theory, and network science allows us to explore core species and interactions embedded within complex dynamics of fish-associated microbiomes.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Purification and characterization of human neural stem and progenitor cells},

author = {Liu, Daniel Dan},

url = {https://www.cell.com/cell/fulltext/S0092-8674(23)00160-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867423001605%3Fshowall%3Dtrue},

doi = {https://doi.org/10.1016/j.cell.2023.02.017},

year  = {2023},

date = {2023-01-01},

abstract = {The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24−THY1−/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1−/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA− bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {A novel fluorogenic reporter substrate for 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2): Application to high-throughput screening for activators to treat Alzheimer's disease},

author = {Visvanathan, Ramya},

url = {https://slas-discovery.org/article/S2472-5552(23)00024-2/fulltext},

doi = {https://doi.org/10.1016/j.slasd.2023.03.003},

year  = {2023},

date = {2023-01-01},

abstract = {A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various “turn-on” fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Discovery of DTX3L inhibitors through a homogeneous FRET-based assay that monitors formation and removal of poly-ubiquitin chains},

author = {Vela-Rodríguez, Carlos},

url = {https://www.biorxiv.org/content/10.1101/2023.03.13.532453v1.article-info},

doi = {https://doi.org/10.1101/2023.03.13.532453},

year  = {2023},

date = {2023-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments},

author = {Gatto, Laurent},

url = {https://www.nature.com/articles/s41592-023-01785-3#Ack1},

doi = {https://doi.org/10.1038/s41592-023-01785-3},

year  = {2023},

date = {2023-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format},

author = {Chen, Yan, et al.},

url = {https://www.protocols.io/view/alkaline-sds-cell-lysis-of-microbes-with-acetone-p-b2raqd2e.html},

doi = {dx.doi.org/10.17504/protocols.io.6qpvr6xjpvmk/v1},

year  = {2023},

date = {2023-01-01},

abstract = {This high-throughput protocol details the steps to extract protein from Gram-negative bacteria, Gram-positive bacteria, or non-filamentous fungi in 96-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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {scONE-seq: A single-cell multi-omics method enables simultaneous dissection of phenotype and genotype heterogeneity from frozen tumors},

author = {Yu, Lei},

url = {https://www.science.org/doi/full/10.1126/sciadv.abp8901},

doi = {https://doi.org/10.1126/sciadv.abp8901},

year  = {2023},

date = {2023-01-01},

abstract = {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.},

keywords = {MANTIS},

pubstate = {published},

tppubtype = {article}

}