Staňurová, Jana A novel screening assay for insulin receptor Journal Article In: 2025. @article{noKey,
title = {A novel screening assay for insulin receptor},
author = {Staňurová, Jana},
url = {https://www.biorxiv.org/content/10.1101/2025.03.20.644157v1.abstract},
doi = {https://doi.org/10.1101/2025.03.20.644157},
year = {2025},
date = {2025-03-20},
abstract = {Insulin is a key hormone in glucose homeostasis. Its lack causes severe health complications and has to be compensated by regular administration of insulin. Despite intense long-lasting research, a more stable substitute has yet to be discovered to alleviate patients' issues. Here we report the development of a novel assay for screening potential insulin analogues based on the recently published method DIANA. Our assay meets the need for a fast, non-radioactive method as a sensitive alternative to the commonly used radioactive immunoassay.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Insulin is a key hormone in glucose homeostasis. Its lack causes severe health complications and has to be compensated by regular administration of insulin. Despite intense long-lasting research, a more stable substitute has yet to be discovered to alleviate patients' issues. Here we report the development of a novel assay for screening potential insulin analogues based on the recently published method DIANA. Our assay meets the need for a fast, non-radioactive method as a sensitive alternative to the commonly used radioactive immunoassay. |
Makhmut, Anuar Deep spatial proteomics of ovarian cancer precursor lesions delineates early disease changes and cell-of-origin signatures Journal Article In: 2025. @article{noKey,
title = {Deep spatial proteomics of ovarian cancer precursor lesions delineates early disease changes and cell-of-origin signatures},
author = {Makhmut, Anuar},
url = {https://www.biorxiv.org/content/10.1101/2025.03.19.643504v1},
doi = {https://doi.org/10.1101/2025.03.19.643504},
year = {2025},
date = {2025-03-19},
abstract = {High-grade serous ovarian cancer (HGSOC) is a devastating disease that is frequently detected at an advanced and incurable stage. Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential. Here, we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma (STIC), the HGSOC precursor lesion, covering more than 10,000 proteins from ultralow input archival tissue. We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level, revealing a similar level of phenotypic and molecular heterogeneity. Using cell-type resolved tissue proteomics, we revealed strong cell-of-origin signatures preserved in STICs and invasive tumors and identified early dysregulated pathways of therapeutic relevance. These include proliferation and DNA damage repair signatures, as well as onco-metabolic changes, such as increased cholesterol biosynthesis. Finally, we uncovered substantial remodeling of the co-evolving tumor microenvironment, affecting approximately one-third of the stromal proteome, and derived a common signature associated with progressive immunosuppression and ECM restructuring. In summary, our study highlights the power of spatially resolved quantitative proteomics to dissect the molecular underpinnings of early carcinogenesis and provides a rich proteomic resource for future biomarker and drug target discovery.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
High-grade serous ovarian cancer (HGSOC) is a devastating disease that is frequently detected at an advanced and incurable stage. Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential. Here, we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma (STIC), the HGSOC precursor lesion, covering more than 10,000 proteins from ultralow input archival tissue. We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level, revealing a similar level of phenotypic and molecular heterogeneity. Using cell-type resolved tissue proteomics, we revealed strong cell-of-origin signatures preserved in STICs and invasive tumors and identified early dysregulated pathways of therapeutic relevance. These include proliferation and DNA damage repair signatures, as well as onco-metabolic changes, such as increased cholesterol biosynthesis. Finally, we uncovered substantial remodeling of the co-evolving tumor microenvironment, affecting approximately one-third of the stromal proteome, and derived a common signature associated with progressive immunosuppression and ECM restructuring. In summary, our study highlights the power of spatially resolved quantitative proteomics to dissect the molecular underpinnings of early carcinogenesis and provides a rich proteomic resource for future biomarker and drug target discovery. |
Escós, Alejandra Disrupted α-ketoglutarate homeostasis trains monocyte-derived macrophages towards M2-like phenotype in long-term treated HIV-infection Journal Article In: 2025. @article{noKey,
title = {Disrupted α-ketoglutarate homeostasis trains monocyte-derived macrophages towards M2-like phenotype in long-term treated HIV-infection},
author = {Escós, Alejandra},
url = {https://www.biorxiv.org/content/10.1101/2025.01.15.633214v1.full},
doi = {https://doi.org/10.1101/2025.01.15.633214},
year = {2025},
date = {2025-03-19},
abstract = {Cells of the myeloid lineage, particularly monocytes and macrophages, play a key role in HIV infection by contributing to viral replication, immune response, and maintaining immune balance during suppressive therapy. We hypothesized that metabolic reprogramming and altered chemokine signaling in people living with HIV (PWH) on long-term antiretroviral therapy (ART) affect monocyte transport and polarization due to ongoing inflammation. Therefore, the present study aimed to identify the mechanism of impaired monocyte/macrophage function in PWH on well-treated ART that can lead to clinical intervention strategies to improve health. Single-cell RNA sequencing, immune-phenotyping, and metabolic modeling identified altered expression of chemokine and metabolite receptors and altered metabolic flux in PWH monocytes that decreased monocyte migration. The plasma secretome revealed a nonclassical inflammatory microenvironment in PWH. Integrative multi-omics and single-cell proteomics of differentiated monocyte-derived macrophages (MDMs) detected metabolic reprogramming orchestrated by α-ketoglutarate (AKG) that affected macrophage function and HIV infection. Increased levels of AKG in plasma were shown to occur in PWH under ART. Therefore, when differentiating MDM with serum from PWH or AKG, macrophage function was found polarized towards an M2-like state. AKG alone was shown to increase CCR5 levels and increase HIV-1 infection in MDM. Here, we utilize systems biology-driven identification and ex vivo assays to show impaired macrophage polarization, due to metabolic training, can leads to a low-grade nonclassical inflammatory environment in well-treated PWH.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Cells of the myeloid lineage, particularly monocytes and macrophages, play a key role in HIV infection by contributing to viral replication, immune response, and maintaining immune balance during suppressive therapy. We hypothesized that metabolic reprogramming and altered chemokine signaling in people living with HIV (PWH) on long-term antiretroviral therapy (ART) affect monocyte transport and polarization due to ongoing inflammation. Therefore, the present study aimed to identify the mechanism of impaired monocyte/macrophage function in PWH on well-treated ART that can lead to clinical intervention strategies to improve health. Single-cell RNA sequencing, immune-phenotyping, and metabolic modeling identified altered expression of chemokine and metabolite receptors and altered metabolic flux in PWH monocytes that decreased monocyte migration. The plasma secretome revealed a nonclassical inflammatory microenvironment in PWH. Integrative multi-omics and single-cell proteomics of differentiated monocyte-derived macrophages (MDMs) detected metabolic reprogramming orchestrated by α-ketoglutarate (AKG) that affected macrophage function and HIV infection. Increased levels of AKG in plasma were shown to occur in PWH under ART. Therefore, when differentiating MDM with serum from PWH or AKG, macrophage function was found polarized towards an M2-like state. AKG alone was shown to increase CCR5 levels and increase HIV-1 infection in MDM. Here, we utilize systems biology-driven identification and ex vivo assays to show impaired macrophage polarization, due to metabolic training, can leads to a low-grade nonclassical inflammatory environment in well-treated PWH. |
Møller, Tenna A. ActinoMation: a literate programming approach for medium-throughput robotic conjugation of Streptomyces spp. Journal Article In: 2025. @article{noKey,
title = {ActinoMation: a literate programming approach for medium-throughput robotic conjugation of Streptomyces spp.},
author = {Møller, Tenna A.},
url = {https://www.sciencedirect.com/science/article/pii/S2405805X25000389},
doi = {https://doi.org/10.1016/j.synbio.2025.03.005},
year = {2025},
date = {2025-03-11},
abstract = {The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds. Advances in molecular engineering tools, such as CRISPR, have provided some access to the metabolic potential of Streptomyces, but efficient genetic engineering of strains is hindered by laborious and slow manual transformation protocols. In this paper, we present a semi-automated medium-throughput workflow for the introduction of recombinant DNA into Streptomyces spp. using the affordable and open-sourced Opentrons (OT-2) robotics platform. To increase the accessibility of the workflow we provide an open-source protocol-creator, ActinoMation. ActinoMation is a literate programming environment using Python in Jupyter Notebook. We validated the method by transforming Streptomyces coelicolor (M1152 and M1146), S. albidoflavus (J1047), and S. venezuelae (DSM40230) with the plasmids pSETGUS and pIJ12551. We demonstrate conjugation efficiencies of 3.33*10-3/0.33% for M1152 with pSETGUS and pIJ12551; 2.96*10-3/0.29%for M1146 with pSETGUS and pIJ12551; 1.21*10-5/0.0012% for J1047 with pSETGUS and 4.70*10-4/0.047% with pIJ12551, and 4.97*10-2/4.97% for DSM40230 with pSETGUS and 6.13*10-2 /6.13% with pIJ12551 with a false positive rate between 8.33% and 54.54%. Automation of the conjugation workflow facilitates a streamlined workflow on a larger scale without any evident loss of conjugation efficiency.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds. Advances in molecular engineering tools, such as CRISPR, have provided some access to the metabolic potential of Streptomyces, but efficient genetic engineering of strains is hindered by laborious and slow manual transformation protocols. In this paper, we present a semi-automated medium-throughput workflow for the introduction of recombinant DNA into Streptomyces spp. using the affordable and open-sourced Opentrons (OT-2) robotics platform. To increase the accessibility of the workflow we provide an open-source protocol-creator, ActinoMation. ActinoMation is a literate programming environment using Python in Jupyter Notebook. We validated the method by transforming Streptomyces coelicolor (M1152 and M1146), S. albidoflavus (J1047), and S. venezuelae (DSM40230) with the plasmids pSETGUS and pIJ12551. We demonstrate conjugation efficiencies of 3.33*10-3/0.33% for M1152 with pSETGUS and pIJ12551; 2.96*10-3/0.29%for M1146 with pSETGUS and pIJ12551; 1.21*10-5/0.0012% for J1047 with pSETGUS and 4.70*10-4/0.047% with pIJ12551, and 4.97*10-2/4.97% for DSM40230 with pSETGUS and 6.13*10-2 /6.13% with pIJ12551 with a false positive rate between 8.33% and 54.54%. Automation of the conjugation workflow facilitates a streamlined workflow on a larger scale without any evident loss of conjugation efficiency. |
Jeevannavar, Aditya Cellular heterogeneity in metabolism and associated microbiome of a non-model phytoflagellate Journal Article In: 2025. @article{noKey,
title = {Cellular heterogeneity in metabolism and associated microbiome of a non-model phytoflagellate},
author = {Jeevannavar, Aditya},
url = {https://academic.oup.com/ismej/advance-article/doi/10.1093/ismejo/wraf046/8064733},
doi = {https://doi.org/10.1093/ismejo/wraf046},
year = {2025},
date = {2025-03-09},
abstract = {Single-cell transcriptomics is a key tool for unravelling metabolism and tissue diversity in model organisms. Its potential for elucidating the ecological roles of microeukaryotes, especially non-model ones, remains largely unexplored. This study employed the Smart-seq2 protocol on Ochromonas triangulata, a microeukaryote lacking a reference genome, showcasing how transcriptional states align with two distinct growth phases: a fast-growing phase and a slow-growing phase. Besides the two expected expression clusters, each corresponding to either growth phase, a third transcriptional state was identified across both growth phases. Metabolic mapping revealed a boost of photosynthetic activity in the fast growth over the slow growth stage, as well as down-regulation trend in pathways associated with ribosome functioning, CO2 fixation, and carbohydrate catabolism characteristic of the third transcriptional state. In addition, carry-over rRNA reads recapitulated the taxonomic identity of the target while revealing distinct bacterial communities, in co-culture with the eukaryote, each associated with distinct transcriptional states. This study underscores single-cell transcriptomics as a powerful tool for characterizing metabolic states in microeukaryotes without a reference genome, offering insights into unknown physiological states and individual-level interactions with different bacterial taxa. This approach holds broad applicability to describe the ecological roles of environmental microeukaryotes, culture-free and reference-free, surpassing alternative methods like metagenomics or metatranscriptomics.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Single-cell transcriptomics is a key tool for unravelling metabolism and tissue diversity in model organisms. Its potential for elucidating the ecological roles of microeukaryotes, especially non-model ones, remains largely unexplored. This study employed the Smart-seq2 protocol on Ochromonas triangulata, a microeukaryote lacking a reference genome, showcasing how transcriptional states align with two distinct growth phases: a fast-growing phase and a slow-growing phase. Besides the two expected expression clusters, each corresponding to either growth phase, a third transcriptional state was identified across both growth phases. Metabolic mapping revealed a boost of photosynthetic activity in the fast growth over the slow growth stage, as well as down-regulation trend in pathways associated with ribosome functioning, CO2 fixation, and carbohydrate catabolism characteristic of the third transcriptional state. In addition, carry-over rRNA reads recapitulated the taxonomic identity of the target while revealing distinct bacterial communities, in co-culture with the eukaryote, each associated with distinct transcriptional states. This study underscores single-cell transcriptomics as a powerful tool for characterizing metabolic states in microeukaryotes without a reference genome, offering insights into unknown physiological states and individual-level interactions with different bacterial taxa. This approach holds broad applicability to describe the ecological roles of environmental microeukaryotes, culture-free and reference-free, surpassing alternative methods like metagenomics or metatranscriptomics. |
Alaviuhkola, Juho Discovery of inhibitors for bacterial Arr enzymes ADP-ribosylating and inactivating rifamycin antibiotics Journal Article In: 2025. @article{noKey,
title = {Discovery of inhibitors for bacterial Arr enzymes ADP-ribosylating and inactivating rifamycin antibiotics},
author = {Alaviuhkola, Juho},
url = {https://www.biorxiv.org/content/10.1101/2025.02.20.639278v1.full},
doi = {https://doi.org/10.1101/2025.02.20.639278},
year = {2025},
date = {2025-02-22},
abstract = {ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD+ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay, which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC50 values below 5 µM against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC50 value of 1.3 µM. We observed structural differences in the NAD+ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD+ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay, which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC50 values below 5 µM against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC50 value of 1.3 µM. We observed structural differences in the NAD+ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds. |
Richardson, Miles SAMPL-seq reveals micron-scale spatial hubs in the human gut microbiome Journal Article In: 2025. @article{noKey,
title = {SAMPL-seq reveals micron-scale spatial hubs in the human gut microbiome},
author = {Richardson, Miles},
url = {https://www.nature.com/articles/s41564-024-01914-4},
doi = {https://doi.org/10.1038/s41564-024-01914-4},
year = {2025},
date = {2025-02-11},
abstract = {The local arrangement of microbes can profoundly impact community assembly, function and stability. However, our understanding of the spatial organization of the human gut microbiome at the micron scale is limited. Here we describe a high-throughput and streamlined method called Split-And-pool Metagenomic Plot-sampling sequencing (SAMPL-seq) to capture spatial co-localization in a complex microbial consortium. The method obtains microbial composition of micron-scale subcommunities through split-and-pool barcoding. SAMPL-seq analysis of the healthy human gut microbiome identified bacterial taxa pairs that consistently co-occurred both over time and across multiple individuals. These co-localized microbes organize into spatially distinct groups or ‘spatial hubs’ dominated by Bacteroidaceae, Ruminococcaceae and Lachnospiraceae families. Using inulin as a dietary perturbation, we observed reversible spatial rearrangement of the gut microbiome where specific taxa form new local partnerships. Spatial metagenomics using SAMPL-seq can unlock insights into microbiomes at the micron scale.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The local arrangement of microbes can profoundly impact community assembly, function and stability. However, our understanding of the spatial organization of the human gut microbiome at the micron scale is limited. Here we describe a high-throughput and streamlined method called Split-And-pool Metagenomic Plot-sampling sequencing (SAMPL-seq) to capture spatial co-localization in a complex microbial consortium. The method obtains microbial composition of micron-scale subcommunities through split-and-pool barcoding. SAMPL-seq analysis of the healthy human gut microbiome identified bacterial taxa pairs that consistently co-occurred both over time and across multiple individuals. These co-localized microbes organize into spatially distinct groups or ‘spatial hubs’ dominated by Bacteroidaceae, Ruminococcaceae and Lachnospiraceae families. Using inulin as a dietary perturbation, we observed reversible spatial rearrangement of the gut microbiome where specific taxa form new local partnerships. Spatial metagenomics using SAMPL-seq can unlock insights into microbiomes at the micron scale. |
Tan, Jeremiah, Bernatsky, Sasha, et al. COVID-19 Breakthrough Infections in Immune-Mediated Inflammatory Diseases: Data from the SUCCEED (Safety and Immunogenicity of COVID-19 Vaccines in Systemic Autoimmune-Mediated Inflammatory Diseases) Study Journal Article In: 2025. @article{noKey,
title = {COVID-19 Breakthrough Infections in Immune-Mediated Inflammatory Diseases: Data from the SUCCEED (Safety and Immunogenicity of COVID-19 Vaccines in Systemic Autoimmune-Mediated Inflammatory Diseases) Study},
author = {Tan, Jeremiah, Bernatsky, Sasha, et al.},
url = {https://www.mdpi.com/2076-393X/13/2/104},
doi = {https://doi.org/10.3390/vaccines13020104},
year = {2025},
date = {2025-01-22},
abstract = {ackground: The Safety and Immunogenicity of COVID-19 Vaccines in Systemic Autoimmune-Mediated Inflammatory Diseases (SUCCEED) study was created to better understand COVID-19 vaccination in immune-mediated inflammatory disease (IMID). Knowing the frequency of COVID-19 breakthrough infections is important, particularly in IMID. Our objective was to assess these events in IMID. Methods: We prospectively studied IMID participants who had received ≥three COVID-19 vaccine doses. Individuals provided saliva samples monthly (September 2022 to August 2023). These were evaluated by polymerase chain reaction (PCR) for SARS-CoV-2. We also assessed antibodies against SARS-CoV-2 (anti-spike, SmT1, receptor binding domain, RBD, and nucleocapsid, NP) based on dried blood spots. Multivariable general estimating equation regression produced odd ratios (OR) for PCR SARS-CoV-2 positivity, related to demographics, immunosuppressives, and antibody levels. Results: Diagnoses included rheumatoid arthritis RA (N = 161, 44% of the total), systemic lupus, psoriatic arthritis, spondylarthritis, vasculitis, systemic sclerosis, and inflammatory bowel disease. Of the 366 participants, most were taking immunosuppressive medication. Of 1266 saliva samples, 56 (5.1%) were positive for SARS-CoV-2 on PCR. Higher anti-SmT1 antibodies were inversely associated with SARS-CoV-2 detection on PCR (adjusted OR 0.66, 95% confidence interval 0.45–0.97). Antibodies to SmT1, RBD, and NP were correlated and thus could not be included in a single model, but when anti-RBD was used in place of anti-SmT1, the results were similar. No other factor (including prior COVID-19 infection) was clearly associated with SARS-CoV-2 detection. Conclusions: This is the first study of SARS-CoV-2 in a large prospective cohort of triple (or more) vaccinated individuals with IMIDs. Anti-SmT1 antibodies appeared to be protective against later SARS-CoV-2 positivity, although recent past infection was not clearly related. This suggests the importance of maintaining robust vaccine-induced immunity through vaccination in IMID.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
ackground: The Safety and Immunogenicity of COVID-19 Vaccines in Systemic Autoimmune-Mediated Inflammatory Diseases (SUCCEED) study was created to better understand COVID-19 vaccination in immune-mediated inflammatory disease (IMID). Knowing the frequency of COVID-19 breakthrough infections is important, particularly in IMID. Our objective was to assess these events in IMID. Methods: We prospectively studied IMID participants who had received ≥three COVID-19 vaccine doses. Individuals provided saliva samples monthly (September 2022 to August 2023). These were evaluated by polymerase chain reaction (PCR) for SARS-CoV-2. We also assessed antibodies against SARS-CoV-2 (anti-spike, SmT1, receptor binding domain, RBD, and nucleocapsid, NP) based on dried blood spots. Multivariable general estimating equation regression produced odd ratios (OR) for PCR SARS-CoV-2 positivity, related to demographics, immunosuppressives, and antibody levels. Results: Diagnoses included rheumatoid arthritis RA (N = 161, 44% of the total), systemic lupus, psoriatic arthritis, spondylarthritis, vasculitis, systemic sclerosis, and inflammatory bowel disease. Of the 366 participants, most were taking immunosuppressive medication. Of 1266 saliva samples, 56 (5.1%) were positive for SARS-CoV-2 on PCR. Higher anti-SmT1 antibodies were inversely associated with SARS-CoV-2 detection on PCR (adjusted OR 0.66, 95% confidence interval 0.45–0.97). Antibodies to SmT1, RBD, and NP were correlated and thus could not be included in a single model, but when anti-RBD was used in place of anti-SmT1, the results were similar. No other factor (including prior COVID-19 infection) was clearly associated with SARS-CoV-2 detection. Conclusions: This is the first study of SARS-CoV-2 in a large prospective cohort of triple (or more) vaccinated individuals with IMIDs. Anti-SmT1 antibodies appeared to be protective against later SARS-CoV-2 positivity, although recent past infection was not clearly related. This suggests the importance of maintaining robust vaccine-induced immunity through vaccination in IMID. |
Duong, Tam Three-dimensional linkage analysis with digital PCR for genome integrity and identity of recombinant adeno-associated virus Journal Article In: 2025. @article{noKey,
title = {Three-dimensional linkage analysis with digital PCR for genome integrity and identity of recombinant adeno-associated virus},
author = {Duong, Tam},
url = {https://www.nature.com/articles/s41598-024-77378-7},
doi = {https://doi.org/10.1038/s41598-024-77378-7},
year = {2025},
date = {2025-01-16},
abstract = {Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product. The conventional single-channel droplet digital PCR (1D ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products which includes not only quantity of viral genome titer but also the quality, distinguishing between partial ones and intact full-length viral genomes with the right GOI. Importantly, due to the random partitioning property of a digital PCR system, the 3D linkage analysis of rAAV viral genome requires a proper mathematical model to identify the true linked DNA molecules (full-length/intact DNA) from the population of false/unlinked DNA molecules (fragmented/partial DNA). We therefore have developed an AAV 3D linkage analysis workflow to characterize genomic integrity and intact titer for rAAV gene therapy products. In this study, we focus on evaluating our 3D linkage mathematical model by performing DNA mixing experiments and a case study using multiple rAAV samples. Particularly, we rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partials, 3 double partials, and 1 full-length genomes. Across all 37 tested scenarios, we validated the accuracy of our workflow’s output for the percentages of 3D linkage by comparing to the known percentages of input DNA. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product. The conventional single-channel droplet digital PCR (1D ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products which includes not only quantity of viral genome titer but also the quality, distinguishing between partial ones and intact full-length viral genomes with the right GOI. Importantly, due to the random partitioning property of a digital PCR system, the 3D linkage analysis of rAAV viral genome requires a proper mathematical model to identify the true linked DNA molecules (full-length/intact DNA) from the population of false/unlinked DNA molecules (fragmented/partial DNA). We therefore have developed an AAV 3D linkage analysis workflow to characterize genomic integrity and intact titer for rAAV gene therapy products. In this study, we focus on evaluating our 3D linkage mathematical model by performing DNA mixing experiments and a case study using multiple rAAV samples. Particularly, we rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partials, 3 double partials, and 1 full-length genomes. Across all 37 tested scenarios, we validated the accuracy of our workflow’s output for the percentages of 3D linkage by comparing to the known percentages of input DNA. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases. |
Usovsky, Mariola Allele-tagged TaqMan® PCR genotyping assays for high-throughput detection of soybean cyst nematode resistance Journal Article In: 2024. @article{noKey,
title = {Allele-tagged TaqMan® PCR genotyping assays for high-throughput detection of soybean cyst nematode resistance},
author = {Usovsky, Mariola},
url = {https://link.springer.com/article/10.1007/s11033-024-10114-6},
doi = {https://doi.org/10.1007/s11033-024-10114-6},
year = {2024},
date = {2024-12-02},
abstract = {Background
Whole genome resequencing (WGRS) platforms provide exceptional fingerprinting of the entire genome but are expensive and less flexible to use as a routine genotyping tool for targeting causal polymorphisms within a germplasm collection or breeding program. Therefore, there has been a continuous effort to develop small-scale genotyping platforms that facilitate robust and quick assessments of the allelic status of causal variants for important traits within soybean breeding programs. The objective was to develop a comprehensive panel of soybean cyst nematode (SCN) resistance TaqMan® assays via selecting the causative genes and analyzing their associated alleles.
Methods
The Soybean Allele Catalog was utilized to investigate WGRS-derived variants which are predicted to cause a change in the amino acid sequence of a gene product. This panel of TaqMan® assays reflects current knowledge about known SCN resistance-causing genes and their associated alleles: GmSNAP18-a and -b, GmSNAP11, GmSHMT08, GmSNAP15, GmNSFRAN07, and GmSNAP02-ins and -del. Developed assays were tested using elite breeding lines and segregating populations. TaqMan assays were compared to other currently available KASP and CAPS assays.
Conclusion
All assays showed excellent allele determination efficiencies. This SCN genotyping assay panel can be utilized as a simplified, accurate and reliable genotyping platform further equipping the updated soybean breeding toolbox.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Background
Whole genome resequencing (WGRS) platforms provide exceptional fingerprinting of the entire genome but are expensive and less flexible to use as a routine genotyping tool for targeting causal polymorphisms within a germplasm collection or breeding program. Therefore, there has been a continuous effort to develop small-scale genotyping platforms that facilitate robust and quick assessments of the allelic status of causal variants for important traits within soybean breeding programs. The objective was to develop a comprehensive panel of soybean cyst nematode (SCN) resistance TaqMan® assays via selecting the causative genes and analyzing their associated alleles.
Methods
The Soybean Allele Catalog was utilized to investigate WGRS-derived variants which are predicted to cause a change in the amino acid sequence of a gene product. This panel of TaqMan® assays reflects current knowledge about known SCN resistance-causing genes and their associated alleles: GmSNAP18-a and -b, GmSNAP11, GmSHMT08, GmSNAP15, GmNSFRAN07, and GmSNAP02-ins and -del. Developed assays were tested using elite breeding lines and segregating populations. TaqMan assays were compared to other currently available KASP and CAPS assays.
Conclusion
All assays showed excellent allele determination efficiencies. This SCN genotyping assay panel can be utilized as a simplified, accurate and reliable genotyping platform further equipping the updated soybean breeding toolbox. |
Chuang, Hsiu-Chun, Ruidong, Li, Li, Li, Kai-Hui, Sun Single-cell sequencing of full-length transcripts and T-cell receptors with automated high-throughput Smart-seq3 Journal Article In: 2024. @article{noKey,
title = {Single-cell sequencing of full-length transcripts and T-cell receptors with automated high-throughput Smart-seq3},
author = {Chuang, Hsiu-Chun, Ruidong, Li, Li, Li, Kai-Hui, Sun},
url = {https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-024-11036-0},
doi = {https://doi.org/10.1186/s12864-024-11036-0},
year = {2024},
date = {2024-11-21},
abstract = {We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples. |
Emrah, Şimşek, Kim, Kiyeri, You, Lingchong A ‘rich-get-richer’ mechanism drives patchy dynamics and resistance evolution in antibiotic-treated bacteria Journal Article In: 2024. @article{noKey,
title = {A ‘rich-get-richer’ mechanism drives patchy dynamics and resistance evolution in antibiotic-treated bacteria},
author = {Emrah, Şimşek, Kim, Kiyeri, You, Lingchong},
url = {https://www.embopress.org/doi/pdf/10.1038/s44320-024-00046-5},
doi = {https://doi.org/10.1038/s44320-024-00046-5},
year = {2024},
date = {2024-06-14},
abstract = {Bacteria in nature often form surface-attached communities that initially comprise distinct subpopulations, or patches. For pathogens, these patches can form at infection sites, persist during antibiotic treatment, and develop into mature biofilms. Evidence suggests that patches can emerge due to heterogeneity in the growth environment and bacterial seeding, as well as cell-cell signaling. However, it is unclear how these factors contribute to patch formation and how patch formation might affect bacterial survival and evolution. Here, we demonstrate that a 'rich-get-richer' mechanism drives patch formation in bacteria exhibiting collective survival (CS) during antibiotic treatment. Modeling predicts that the seeding heterogeneity of these bacteria is amplified by local CS and global resource competition, leading to patch formation. Increasing the dose of a non-eradicating antibiotic treatment increases the degree of patchiness. Experimentally, we first demonstrated the mechanism using engineered Escherichia coli and then demonstrated its applicability to a pathogen, Pseudomonas aeruginosa. We further showed that the formation of P. aeruginosa patches promoted the evolution of antibiotic resistance. Our work provides new insights into population dynamics and resistance evolution during surface-attached bacterial growth.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Bacteria in nature often form surface-attached communities that initially comprise distinct subpopulations, or patches. For pathogens, these patches can form at infection sites, persist during antibiotic treatment, and develop into mature biofilms. Evidence suggests that patches can emerge due to heterogeneity in the growth environment and bacterial seeding, as well as cell-cell signaling. However, it is unclear how these factors contribute to patch formation and how patch formation might affect bacterial survival and evolution. Here, we demonstrate that a 'rich-get-richer' mechanism drives patch formation in bacteria exhibiting collective survival (CS) during antibiotic treatment. Modeling predicts that the seeding heterogeneity of these bacteria is amplified by local CS and global resource competition, leading to patch formation. Increasing the dose of a non-eradicating antibiotic treatment increases the degree of patchiness. Experimentally, we first demonstrated the mechanism using engineered Escherichia coli and then demonstrated its applicability to a pathogen, Pseudomonas aeruginosa. We further showed that the formation of P. aeruginosa patches promoted the evolution of antibiotic resistance. Our work provides new insights into population dynamics and resistance evolution during surface-attached bacterial growth. |
Zukas, Kieran Rapid high-throughput method for investigating physiological regulation of neutrophil extracellular trap formation Journal Article In: 2024. @article{noKey,
title = {Rapid high-throughput method for investigating physiological regulation of neutrophil extracellular trap formation},
author = {Zukas, Kieran},
url = {https://www.sciencedirect.com/science/article/pii/S1538783624003209},
doi = {https://doi.org/10.1016/j.jtha.2024.05.028},
year = {2024},
date = {2024-06-10},
abstract = {Background
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.
Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.
Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.
Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Background
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.
Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.
Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.
Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment. |
Tan, Tao Protocol for generation of and high-throughput drug testing with patient-derived colorectal cancer organoids Journal Article In: 2024. @article{noKey,
title = {Protocol for generation of and high-throughput drug testing with patient-derived colorectal cancer organoids},
author = {Tan, Tao},
url = {https://www.sciencedirect.com/science/article/pii/S2666166724002557},
doi = {https://doi.org/10.1016/j.xpro.2024.103090},
year = {2024},
date = {2024-05-28},
abstract = {Drug sensitivity testing of patient-derived tumor organoids (PDTOs) is a promising tool for personalizing cancer treatment. Here, we present a protocol for generation of and high-throughput drug testing with PDTOs. We describe detailed steps for PDTO establishment from colorectal cancer tissues, preparation of PDTOs for high-throughput drug testing, and quantification of drug testing results using image analysis. This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies, along with exploring the activity of new agents, for translational research.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Drug sensitivity testing of patient-derived tumor organoids (PDTOs) is a promising tool for personalizing cancer treatment. Here, we present a protocol for generation of and high-throughput drug testing with PDTOs. We describe detailed steps for PDTO establishment from colorectal cancer tissues, preparation of PDTOs for high-throughput drug testing, and quantification of drug testing results using image analysis. This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies, along with exploring the activity of new agents, for translational research. |
Huertas, Luis Hernandez CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of the maternal-to-zygotic transition in teleosts Journal Article In: 2024. @article{noKey,
title = {CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of the maternal-to-zygotic transition in teleosts},
author = {Huertas, Luis Hernandez},
url = {https://www.biorxiv.org/content/10.1101/2024.05.22.595167v1.full},
doi = {https://doi.org/10.1101/2024.05.22.595167},
year = {2024},
date = {2024-05-23},
abstract = {The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation. |
Williams, Jonathan David Dynamic Clinical Assay Pipeline for Detecting a Virus Journal Article In: 2024. @article{noKey,
title = {Dynamic Clinical Assay Pipeline for Detecting a Virus},
author = {Williams, Jonathan David},
url = {https://patents.google.com/patent/US20240141447A1/en},
doi = {US20240141447A1},
year = {2024},
date = {2024-05-02},
abstract = {Disclosed herein are methods and systems comprising obtaining nucleic acid from a sample that was obtained from a subject; capturing and amplifying a target molecule in the nucleic acid using a molecular inversion probe under hybridization conditions; ligating an adapter to create a circular molecule; sequencing the circular molecule to obtain sequence reads; generating a sequencing file comprising the sequence reads of each molecule and a position of each sequence read in a reference genome of a virus; and generating a reporting file for the subject comprising a predicted lineage of the virus in the sample.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Disclosed herein are methods and systems comprising obtaining nucleic acid from a sample that was obtained from a subject; capturing and amplifying a target molecule in the nucleic acid using a molecular inversion probe under hybridization conditions; ligating an adapter to create a circular molecule; sequencing the circular molecule to obtain sequence reads; generating a sequencing file comprising the sequence reads of each molecule and a position of each sequence read in a reference genome of a virus; and generating a reporting file for the subject comprising a predicted lineage of the virus in the sample. |
Steinhauser, Sebastian The transcription factor ZNF469 regulates collagen production in liver fibrosis Journal Article In: 2024. @article{noKey,
title = {The transcription factor ZNF469 regulates collagen production in liver fibrosis},
author = {Steinhauser, Sebastian},
url = {https://www.biorxiv.org/content/10.1101/2024.04.25.591188v1},
doi = {https://doi.org/10.1101/2024.04.25.591188},
year = {2024},
date = {2024-04-25},
abstract = {Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis. |
Cieślak, Marcin Machine learning accelerates pharmacophore-based virtual screening of MAO inhibitors Journal Article In: 2024. @article{noKey,
title = {Machine learning accelerates pharmacophore-based virtual screening of MAO inhibitors},
author = {Cieślak, Marcin},
url = {https://www.nature.com/articles/s41598-024-58122-7},
doi = {https://doi.org/10.1038/s41598-024-58122-7},
year = {2024},
date = {2024-04-08},
abstract = {Nowadays, an efficient and robust virtual screening procedure is crucial in the drug discovery process, especially when performed on large and chemically diverse databases. Virtual screening methods, like molecular docking and classic QSAR models, are limited in their ability to handle vast numbers of compounds and to learn from scarce data, respectively. In this study, we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures. The developed protocol yielded 1000 times faster binding energy predictions than classical docking-based screening. The proposed predictive model learns from docking results, allowing users to choose their preferred docking software without relying on insufficient and incoherent experimental activity data. The methodology described employs multiple types of molecular fingerprints and descriptors to construct an ensemble model that further reduces prediction errors and is capable of delivering highly precise docking score values for monoamine oxidase ligands, enabling faster identification of promising compounds. An extensive pharmacophore-constrained screening of the ZINC database resulted in a selection of 24 compounds that were synthesized and evaluated for their biological activity. A preliminary screen discovered weak inhibitors of MAO-A with a percentage efficiency index close to a known drug at the lowest tested concentration. The approach presented here can be successfully applied to other biological targets as target-specific knowledge is not incorporated at the screening phase.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Nowadays, an efficient and robust virtual screening procedure is crucial in the drug discovery process, especially when performed on large and chemically diverse databases. Virtual screening methods, like molecular docking and classic QSAR models, are limited in their ability to handle vast numbers of compounds and to learn from scarce data, respectively. In this study, we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures. The developed protocol yielded 1000 times faster binding energy predictions than classical docking-based screening. The proposed predictive model learns from docking results, allowing users to choose their preferred docking software without relying on insufficient and incoherent experimental activity data. The methodology described employs multiple types of molecular fingerprints and descriptors to construct an ensemble model that further reduces prediction errors and is capable of delivering highly precise docking score values for monoamine oxidase ligands, enabling faster identification of promising compounds. An extensive pharmacophore-constrained screening of the ZINC database resulted in a selection of 24 compounds that were synthesized and evaluated for their biological activity. A preliminary screen discovered weak inhibitors of MAO-A with a percentage efficiency index close to a known drug at the lowest tested concentration. The approach presented here can be successfully applied to other biological targets as target-specific knowledge is not incorporated at the screening phase. |
Visvanathan, Ramya A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes Journal Article In: 2024. @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 = {2024-03-29},
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}
}
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. |
Baglaenko, Yuriy Defining the function of disease variants with CRISPR editing and multimodal single cell sequencing Journal Article In: 2024. @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 = {2024-03-29},
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}
}
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. |
