An R-loop is a triple-stranded nucleic acid structure consisting of a DNA–RNA hybrid and a displaced single-stranded DNA. R-loops are associated with diverse biological reactions, such as immune responses and gene regulation, and dysregulated R-loops can cause genomic instability and replication stress. Therefore, investigating the formation, regulation, and elimination of R-loops is important for understanding the molecular mechanisms underlying biological processes and diseases related to R-loops. Existing research has primarily focused on R-loop detection. In this chapter, we introduce a variety of biochemical and biophysical techniques for R-loop sensing and visualization both in vivo and in vitro, including single-molecule imaging. These methods can be used to investigate molecular mechanisms underlying R-loop search and identification.
This collection of processed data accompanies the study: Yeh, Aguirre, Laveroni et al.Mapping spatial organization in ovarian cancer uncovers drivers of immune evasion. (2023). Unpublished. This collection of processed data is purposed for easy access and computing directly in the R programming environment. All files are provided in the form of RObjects (extension .rds). Data collected and processed for the first time as a part of this study include: de-identified clinical annotations for a cohort of 60 patients diagnosed with tubo-ovarian high grade serous carcinoma (HGSC), morphology annotations from clinical images (Hematoxylin and Eosin), targeted genomic data, single cell spatial transcriptomics on 95 HGSC tumors, CRISPR-screen data in ovarian cancer cells in co-culture with T and Natural Killer (NK) cells, and Perturb-Seq data in ovarian cancer cells co-cultured with NK cells. Processed data from published journal articles include scRNA-Seq data downloaded from Synapse (syn33521743), Gene Expression Omnnibus (GSE118828, GSE173682, GSE147082, GSE154600, GSE146026). This following table provides a description of the files contained in this collection. File Object Type in R Description 01_Clinical.rds data.frame Clinical annotations of patients in above study. 02_ClinicalDataColumnKey.rds data.frame Key for Column IDs in 01_Clinical.rds 03_CNA.rds matrix Patient x Gene matrix of copy number alterations. 04_SMI_data.rds list Single cell spatial transcriptomics data collected on CosMx Single Molecule Imaging Platform (Discovery Dataset). This list contains the raw counts data in the field "cd" and transcripts per million data in "tpm". All meta data on the cell-level including cell.types is stored in this list. 05_Xenium_data.rds list Single cell spatial transcriptomics data collected on 10X Xenium Platform (Validation Dataset 1). This list contains the raw counts data in the field "cd" and transcripts per million data in "tpm". All meta data on the cell-level including cell.types is stored in this list. 06_MERFISH_data.rds list Single cell spatial transcriptomics data collected on Vizgen's Multiplexed Error Robust Fluorescent in situ Hybridization Platform (Validation Dataset 2). This list contains the raw counts data in the field "cd" and transcripts per million data in "tpm". All meta data on the cell-level including cell.types is stored in this list. 07_scRNA_Geistliner_r.rds list Single cell RNA-Seq data downloaded from GEO (GSE154600), processed to match the data structure of the spatial transcriptomics data described for 04_SMI_data.rds, 05_Xenium_data.rds, 06_MERFISH_data.rds. 08_scRNA_Olalekan_r.rds list Single cell RNA-Seq data downloaded from GEO (GSE147082), processed to match the data structure of the spatial transcriptomics data described for 04_SMI_data.rds, 05_Xenium_data.rds, 06_MERFISH_data.rds. 09_scRNA_Qian_r.rds list Single cell RNA-Seq data downloaded from https://lambrechtslab.sites.vib.be/en/data-access, processed to match the data structure of the spatial transcriptomics data described for 04_SMI_data.rds, 05_Xenium_data.rds, 06_MERFISH_data.rds. 10_scRNA_Regner_r.rds list Single cell RNA-Seq data downloaded from GEO (GSE173682), processed to match the data structure of the spatial transcriptomics data described for 04_SMI_data.rds, 05_Xenium_data.rds, 06_MERFISH_data.rds. 11_scRNA_Shih_r.rds list Single cell RNA-Seq data downloaded from GEO (GSE118828), processed to match the data structure of the spatial transcriptomics data described for 04_SMI_data.rds, 05_Xenium_data.rds, 06_MERFISH_data.rds. 12_scRNA_VazquezGarcia_r.rds list Single cell RNA-Seq data downloaded from Synapse (syn33521743), processed to match the data structure of the spatial transcriptomics data described for 04_SMI_data.rds, 05_Xenium_data.rds, 06_MERFISH_data.rds. 13_StromalMorphology.rds data.frame Tissue morphology annotations made by a board-certified gynecological pathologist of each sample in the SMI (Discovery) dataset 14_CRISPR.rds data.frame Z-scores of ovarian cancer cell line resistance to T and NK cell mediated killing based as a function of genetic perturbations via CRISPR. 15_PerturbSeq_TYKnuNK.rds list Perturb-Seq data of ovarian cancer cells under co-culture with NK cells. This list contains the raw counts data in the field "cd" and transcripts per million data in "tpm". All meta data on the cell-level is stored in this list.
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease resulting from motor neuron degeneration that causes muscle weakness, paralysis, and eventually respiratory failure. We investigated whether recombinant adeno-associated virus encoding human hepatocyte growth factor (rAAV-HGF) could generate beneficial effects in two mouse models with neuromuscular problems when intrathecally delivered to the subarachnoid space. We chose AAV serotype 1 (rAAV1) based on the expression levels and distribution of HGF protein in the lumbar spinal cord (LSC). After a single intrathecal (IT) injection of rAAV1-HGF, the protein level of HGF in the LSC peaked on day 14 and thereafter gradually decreased over the next 14 weeks. rAAV1-HGF was initially tested in the mouse nerve crush model. IT injection of rAAV1-HGF improved mouse hindlimb strength and rotarod performance, while histological analyses showed that the length of regenerated axons was increased and the structure of the neuromuscular junction (NMJ) was restored. rAAV1-HGF was also evaluated in the SOD1-G93A transgenic (TG) mouse model. Again, rAAV1-HGF not only improved motor performance but also increased the survival rate. Moreover, the number and diameter of spinal motor neurons (SMNs) were increased, and the shape of the NMJs restored. Data from in vitro motor cortical culture experiments indicated that treatment with recombinant HGF protein (rHGF) increased the axon length of corticospinal motor neurons (CSMNs). When cultures were treated with an ERK inhibitor, the effects of HGF on axon elongation, protein aggregation, and oxidative stress were suppressed, indicating that ERK phosphorylation played an important role(s). Taken together, our results suggested that HGF might play an important role(s) in delaying disease progression in the SOD1-G93A TG mouse model by reducing oxidative stress through the control of ERK phosphorylation.
Abstract TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.
The drivers of immune evasion are not entirely clear, limiting the success of cancer immunotherapies. Here we applied single-cell spatial and perturbational transcriptomics to delineate immune evasion in high-grade serous tubo-ovarian cancer. To this end, we first mapped the spatial organization of high-grade serous tubo-ovarian cancer by profiling more than 2.5 million cells in situ in 130 tumors from 94 patients. This revealed a malignant cell state that reflects tumor genetics and is predictive of T cell and natural killer cell infiltration levels and response to immune checkpoint blockade. We then performed Perturb-seq screens and identified genetic perturbations—including knockout of PTPN1 and ACTR8—that trigger this malignant cell state. Finally, we show that these perturbations, as well as a PTPN1/PTPN2 inhibitor, sensitize ovarian cancer cells to T cell and natural killer cell cytotoxicity, as predicted. This study thus identifies ways to study and target immune evasion by linking genetic variation, cell-state regulators and spatial biology. Here the authors provide a resource for ovarian cancer combining spatial transcriptomics, genomics, CRISPR Perturb-seq screens and in silico methods to focus on T cells and natural killer cells in the tumor and their role in immune evasion.
SUMMARY Immune exclusion and evasion are central barriers to the success of immunotherapies and cell therapies in solid tumors. Here we applied single cell spatial and perturbational transcriptomics alongside clinical, histological, and genomic profiling to elucidate immune exclusion and evasion in high-grade serous tubo-ovarian cancer (HGSC). Using high-plex spatial transcriptomics we profiled more than 1.3 million cells from 95 tumors and 60 patients, revealing generalizable principles in HGSC tumor tissue organization. Our data demonstrates that effector T cells resist stroma-mediated trapping and sequestration. However, upon infiltration into the tumor, T cells, as well as Natural Killer (NK) cells, preferentially co-localize only with a subset of malignant cells that manifest a distinct transcriptional cell state. The latter consists of dozens of co-regulated genes and is repressed under various copy number alterations. Performing CRISPR Perturb-seq screens in ovarian cancer cells, we identified functionally diverse genetic perturbations – including knockout of the insulin sensing repressor PTPN1 and the epigenetic regulator ACTR8 – that de-repress the proposed immunogenic malignant cell state identified in patients and indeed sensitize ovarian cancer cells to T cell and NK cell cytotoxicity. Taken together, our study uncovered a profound connection between somatic genetic aberrations, malignant cell transcriptional dysregulation, and immune evasion at the cellular and tissue level, allowing us to identify targets that reprogram malignant cell states as an avenue to unleash anti-tumor immune responses.
We developed a prototype of optical imaging-based pointof-care (POC) devices that can detect CD4+ T-lymphocytes in human blood for HIV/AIDS monitoring. The proposed portable cell-counting system, Helios CD4, can acquire sample images and analyze particles or cells automatically, by using a simple imaging module and a sample cartridge with a three-dimensional (3D) helical mini-channel. This device has advantages over the existing devices because of its small size and simple scanning mechanism. A performance evaluation was conducted by comparing the cell count obtained using Helios with that obtained using PIMA, one of the most widely used POC CD4+ cell counter.
Abstract Background Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of motor neurons (MNs), leading to paralysis, respiratory failure and death within 2–5 years of diagnosis. The exact mechanisms of sporadic ALS, which comprises 90% of all cases, remain unknown. In familial ALS, mutations in superoxide dismutase (SOD1) cause 10% of cases. Methods ALS patient-derived human-induced pluripotent stem cells (ALS hiPSCs, harboring the SOD1 AV4 mutation), were differentiated to MNs (ALS-MNs). The neuroprotective effects of conditioned medium (CM) of hESCs (H9), wt hiPSCs (WTC-11) and the ALS iPSCs, on MN apoptosis and viability, formation and maintenance of neurites, mitochondrial activity and expression of inflammatory genes, were examined. For in vivo studies, 200 μl of CM from the ALS iPSCs (CS07 and CS053) was injected subcutaneously into the ALS model mice (transgenic for the human SOD1 G93A mutation). Animal agility and strength, muscle innervation and mass, neurological score, onset of paralysis and lifespan of the ALS mice were assayed. After observing significant disease-modifying effects, the CM was characterized biochemically by fractionation, comparative proteomics, and epigenetic screens for the dependence on pluripotency. CM of fibroblasts that were differentiated from the wt hiPSCs lacked any neuroprotective activity and was used as a negative control throughout the studies. Results The secretome of PSCs including the ALS patient iPSCs was neuroprotective in the H 2 O 2 model. In the model with pathogenic SOD1 mutation, ALS iPSC-CM attenuated all examined hallmarks of ALS pathology, rescued human ALS-MNs from denervation and death, restored mitochondrial health, and reduced the expression of inflammatory genes. The ALS iPSC-CM also improved neuro-muscular health and function, and delayed paralysis and morbidity in ALS mice. Compared side by side, cyclosporine (CsA), a mitochondrial membrane blocker that prevents the leakage of mitochondrial DNA, failed to avert the death of ALS-MNs, although CsA and ALS iPSC-CM equally stabilized MN mitochondria and attenuated inflammatory genes. Biochemical characterization, comparative proteomics, and epigenetic screen all suggested that it was the interactome of several key proteins from different fractions of PSC-CM that delivered the multifaceted neuroprotection. Conclusions This work introduces and mechanistically characterizes a new biologic for treating ALS and other complex neurodegenerative diseases.
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