Inherited deficiency of stress granule ZNFX1 in patients with monocytosis and mycobacterial disease.
French Institute of Health and Medical Research1, University of Paris2, Rockefeller University3, King Abdulaziz Medical City4, King Saud bin Abdulaziz University for Health Sciences5, Qatar Airways6, Free University of Brussels7, Icahn School of Medicine at Mount Sinai8, Agency for Science, Technology and Research9, King Saud University10, Khalifa University11, Shahid Beheshti University of Medical Sciences and Health Services12
13 Apr 2021-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 118, Iss: 15
TL;DR: In this paper, the authors found that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.
Abstract: Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette-Guerin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.
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TL;DR: In this paper , the authors report the updated classification of inborn errors of immunity, compiled by the International Union of Immunological Societies Expert Committee, which is designed to serve as a resource for immunologists and geneticists pursuing the molecular diagnosis of individuals with heritable immunological disorders.
Abstract: We report the updated classification of inborn errors of immunity, compiled by the International Union of Immunological Societies Expert Committee. This report documents the key clinical and laboratory features of 55 novel monogenic gene defects, and 1 phenocopy due to autoantibodies, that have either been discovered since the previous update (published January 2020) or were characterized earlier but have since been confirmed or expanded in subsequent studies. While variants in additional genes associated with immune diseases have been reported in the literature, this update includes only those that the committee assessed that reached the necessary threshold to represent novel inborn errors of immunity. There are now a total of 485 inborn errors of immunity. These advances in discovering the genetic causes of human immune diseases continue to significantly further our understanding of molecular, cellular, and immunological mechanisms of disease pathogenesis, thereby simultaneously enhancing immunological knowledge and improving patient diagnosis and management. This report is designed to serve as a resource for immunologists and geneticists pursuing the molecular diagnosis of individuals with heritable immunological disorders and for the scientific dissection of cellular and molecular mechanisms underlying monogenic and related human immune diseases.
185 citations
TL;DR: In this article, the authors demonstrate that RNF213 E3 activity is dependent on ATP binding, rather than ATP hydrolysis, and is particularly responsive to the ATP/ADP/AMP ratio.
Abstract: RNF213 is a giant E3 ubiquitin ligase and a major susceptibility factor of Moyamoya disease, a cerebrovascular disorder that can result in stroke or death. In the cell, RNF213 is involved in lipid droplet formation, lipotoxicity, hypoxia, and NF-κB signaling, but its exact function in these processes is unclear. Structural characterization has revealed the presence of a dynein- like ATPase module and an unprecedented but poorly understood E3 module. Here, we demonstrate that RNF213 E3 activity is dependent on ATP binding, rather than ATP hydrolysis, and is particularly responsive to the ATP/ADP/AMP ratio. Biochemical and activity-based probe analyses identify a non-canonical zinc finger domain as the E3 active site, which utilizes the strictly conserved Cys4462, not involved in zinc coordination, as the reactive nucleophile. The cryo-EM structure of the trapped RNF213:E2∼Ub intermediate reveals RNF213 C-terminal domain as the E2 docking site, which positions the ubiquitin-loaded E2 proximal to the catalytic zinc finger, facilitating nucleophilic attack of Cys4462 on the E2∼Ub thioester. Our findings show that RNF213 represents an undescribed type of a transthiolation E3 enzyme and is regulated by adenine nucleotide concentration via its ATPase core, possibly allowing it to react to changing metabolic conditions in the cell.
17 citations
TL;DR: In this paper, the authors reviewed recent progress in the study of inborn errors of immunity underlying mycobacterial diseases, including the impairment of immunity mediated by interferon gamma (IFN-γ).
13 citations
TL;DR: In this paper , the authors show that RNA mediated interference leads to heritable changes in the distribution of nascent and mature transcripts that correlate with two parallel sRNA amplification loops, one targeting nascent transcripts and the other targeting mature transcripts and concentrates them in perinuclear condensates.
Abstract: Abstract RNA-mediated interference (RNAi) is a conserved mechanism that uses small RNAs (sRNAs) to silence gene expression. In the Caenorhabditis elegans germline, transcripts targeted by sRNAs are used as templates for sRNA amplification to propagate silencing into the next generation. Here we show that RNAi leads to heritable changes in the distribution of nascent and mature transcripts that correlate with two parallel sRNA amplification loops. The first loop, dependent on the nuclear Argonaute HRDE-1, targets nascent transcripts and reduces but does not eliminate productive transcription at the locus. The second loop, dependent on the conserved helicase ZNFX-1, targets mature transcripts and concentrates them in perinuclear condensates. ZNFX-1 interacts with sRNA-targeted transcripts that have acquired poly(UG) tails and is required to sustain pUGylation and robust sRNA amplification in the inheriting generation. By maintaining a pool of transcripts for amplification, ZNFX-1 prevents premature extinction of the RNAi response and extends silencing into the next generation.
12 citations
TL;DR: In this article , a detailed protocol for the mass production of human induced pluripotent stem cells (iPSC)-derived macrophages was proposed, which is straightforward, robust and characterized by the differentiation of primed iPSC aggregates into myeloid-cell-forming-complex intermediates by means of a minimal cytokine cocktail.
Abstract: Macrophages derived from human induced pluripotent stem cells (iPSCs) have the potential to enable the development of cell-based therapies for numerous disease conditions. We here provide a detailed protocol for the mass production of iPSC-derived macrophages (iPSC-Mac) in scalable suspension culture on an orbital shaker or in stirred-tank bioreactors (STBRs). This strategy is straightforward, robust and characterized by the differentiation of primed iPSC aggregates into 'myeloid-cell-forming-complex' intermediates by means of a minimal cytokine cocktail. In contrast to the 'batch-like differentiation approaches' established for other iPSC-derived lineages, myeloid-cell-forming-complex-intermediates are stably maintained in suspension culture and continuously generate functional and highly pure iPSC-Mac. Employing a culture volume of 120 ml in the STBR platform, ~1-4 × 107 iPSC-Mac can be harvested at weekly intervals for several months. The STBR technology allows for real-time monitoring of crucial process parameters such as biomass, pH, dissolved oxygen, and nutrition levels; the system also promotes systematic process development, optimization and linear upscaling. The process duration, from the expansion of iPSC until the first iPSC-Mac harvest, is 28 d. Successful application of the protocol requires expertise in pluripotent stem cell culture, differentiation and analytical methods, such as flow cytometry. Fundamental know-how in biotechnology is also advantageous to run the process in the STBR platform. The continuous, scalable production of well-defined iPSC-Mac populations is highly relevant to various fields, ranging from developmental biology, immunology and cell therapies to industrial applications for drug safety and discovery.
12 citations
References
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TL;DR: SAMtools as discussed by the authors implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.
Abstract: Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.
Availability: http://samtools.sourceforge.net
Contact: [email protected]
45,957 citations
TL;DR: Burrows-Wheeler Alignment tool (BWA) is implemented, a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps.
Abstract: Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals.
Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ~10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package.
Availability: http://maq.sourceforge.net
Contact: [email protected]
43,862 citations
TL;DR: The consensus clustering (CC) method provides quantitative and visual stability evidence for estimating the number of unsupervised classes in a dataset and ConsensusClusterPlus implements the CC method in R and extends it with new functionality and visualizations including item tracking, item- Consensus and cluster-consensus plots.
Abstract: Summary: Unsupervised class discovery is a highly useful technique in cancer research, where intrinsic groups sharing biological characteristics may exist but are unknown. The consensus clustering (CC) method provides quantitative and visual stability evidence for estimating the number of unsupervised classes in a dataset. ConsensusClusterPlus implements the CC method in R and extends it with new functionality and visualizations including item tracking, item-consensus and cluster-consensus plots. These new features provide users with detailed information that enable more specific decisions in unsupervised class discovery.
Availability: ConsensusClusterPlus is open source software, written in R, under GPL-2, and available through the Bioconductor project (http://www.bioconductor.org/).
Contact: ude.cnu.dem@srekliwm
Supplementary Information: Supplementary data are available at Bioinformatics online.
2,694 citations
TL;DR: Harmony, for the integration of single-cell transcriptomic data, identifies broad and fine-grained populations, scales to large datasets, and can integrate sequencing- and imaging-based data.
Abstract: The emerging diversity of single-cell RNA-seq datasets allows for the full transcriptional characterization of cell types across a wide variety of biological and clinical conditions. However, it is challenging to analyze them together, particularly when datasets are assayed with different technologies, because biological and technical differences are interspersed. We present Harmony (
https://github.com/immunogenomics/harmony
), an algorithm that projects cells into a shared embedding in which cells group by cell type rather than dataset-specific conditions. Harmony simultaneously accounts for multiple experimental and biological factors. In six analyses, we demonstrate the superior performance of Harmony to previously published algorithms while requiring fewer computational resources. Harmony enables the integration of ~106 cells on a personal computer. We apply Harmony to peripheral blood mononuclear cells from datasets with large experimental differences, five studies of pancreatic islet cells, mouse embryogenesis datasets and the integration of scRNA-seq with spatial transcriptomics data. Harmony, for the integration of single-cell transcriptomic data, identifies broad and fine-grained populations, scales to large datasets, and can integrate sequencing- and imaging-based data.
2,459 citations
TL;DR: The mechanisms that control monocyte trafficking under homeostatic, infectious and inflammatory conditions are being unravelled and are the focus of this Review.
Abstract: Monocytes originate from progenitors in the bone marrow and traffic via the bloodstream to peripheral tissues. During both homeostasis and inflammation, circulating monocytes leave the bloodstream and migrate into tissues where, following conditioning by local growth factors, pro-inflammatory cytokines and microbial products, they differentiate into macrophage or dendritic cell populations. Recruitment of monocytes is essential for effective control and clearance of viral, bacterial, fungal and protozoal infections, but recruited monocytes also contribute to the pathogenesis of inflammatory and degenerative diseases. The mechanisms that control monocyte trafficking under homeostatic, infectious and inflammatory conditions are being unravelled and are the focus of this Review.
2,309 citations