Biology subjects

Shivram, H.

Publications and source records attributed to Shivram, H..

7 recordsLinked to original sources

bioRxiv2024

G-CSF and IL-6 drive myeloid dysregulation during severe viral infection

Dysregulated myeloid states are associated with disease severity in both sepsis and COVID-19. However, their relevance in non-COVID-19 viral infection, the factors driving their induction, and their role in tissue injury remain poorly understood. We performed a meta-analysis of 1,622,180 myeloid cells from 890 COVID-19 or sepsis patients and controls across 19 published blood scRNA-seq datasets, which revealed severity-associated gene programs in both neutrophils and monocytes pointing to emergency myelopoiesis (EM). Using published bulk transcriptional data from 562 individuals with non-COVID-19 viral disease, we show that these signatures are similarly upregulated during severe influenza and RSV infection. Analysis of transcriptional and proteomic responses in tocilizumab-treated COVID-19 patients show that IL-6 signaling blockade results in a partial reduction of EM signatures and a compensatory increase in the growth factor G-CSF. Using a cellular model of human myelopoiesis, we show that both IL-6 and G-CSF stimulate the production of myeloid cells that express EM signatures in vitro. Using a mouse model of severe influenza infection, we demonstrate the effect of IL-6 and G-CSF signaling blockade on EM-associated myeloid cells, and highlight the opposing effects of EM-induced neutrophils and monocytes on tissue injury. Our study demonstrates the link between systemic cytokines and myeloid dysregulation during severe infection in humans, and highlights the cooperative role of IL-6 and G-CSF signaling in driving infection-induced myelopoiesis.

immunology↗

bioRxiv2022

Tocilizumab treatment leads to early resolution of myeloid dysfunction and lymphopenia in patients hospitalized with COVID-19

High interleukin (IL)-6 levels are associated with more severe clinical manifestations in patients hospitalized with COVID-19, but the complex role of IL-6 in antiviral and inflammatory processes has made it difficult to decipher its involvement in the disease. IL-6 receptor blockade by tocilizumab (anti-IL6R; Actemra) is used globally for the treatment of severe COVID-19, yet a molecular understanding of the therapeutic benefit remains unclear. We characterized the immune profile and identified cellular and molecular pathways directly modified by tocilizumab in peripheral blood samples collected from patients enrolled in the COVACTA study, a phase 3, randomized, double-blind, placebo-controlled trial that assessed the efficacy and safety of tocilizumab in hospitalized patients with severe COVID-19 pneumonia. We identified factors predicting disease severity and clinical outcomes, including markers of inflammation, lymphopenia, myeloid dysregulation, and organ injury. Proteomic analysis confirmed a pharmacodynamic effect for tocilizumab in addition to identifying novel pharmacodynamic biomarkers. Transcriptomic analysis revealed that tocilizumab treatment leads to faster resolution of lymphopenia and myeloid dysregulation associated with severe COVID-19, indicating greater anti-inflammatory activity relative to standard of care and potentially leading to faster recovery in patients hospitalized with COVID-19. One sentence summaryInterleukin-6 receptor blockade with tocilizumab accelerated resolution of myeloid dysfunction and lymphopenia in patients hospitalized with COVID-19

immunology↗

bioRxiv2021

Diverse ATPase proteins in mobilomes constitute a large potential sink for prokaryotic host ATP

Prokaryote mobilome genomes rely on host machineries for survival and replication. Given that mobile genetic elements (MGEs) derive their energy from host cells, we investigated the diversity of ATP-utilizing proteins in MGE genomes to determine whether they might be associated with proteins that could suppress related host proteins that consume host energy. A comprehensive search of 353 huge phage genomes revealed that up to 9% of the proteins have ATPase domains. For example, ATPase proteins constitute ~3% of the genomes of Lak phages with ~550 kbp genomes that occur in the microbiomes of humans and other animals. Statistical analysis shows the number of ATPase proteins increases linearly with genome length, consistent with a large sink for host ATP during replication of megaphages. Using metagenomic data from diverse environments, we found 505 mobilome proteins with ATPase domains fused to diverse functional domains. Among these composite ATPase proteins, 61.6% have known functional domains that could contribute to host energy diversion during the mobilome life cycle. As many have domains that are known to interact with nucleic acids and proteins, we infer that numerous ATPase proteins are used during replication and for protection from host immune systems. We found a set of uncharacterized ATPase proteins with nuclease and protease activities, displaying unique domain architectures that are energy intensive based on the presence of multiple ATPase domains. In many cases, these composite ATPase proteins genomically co-localize with small proteins in genomic contexts that are reminiscent of toxin-antitoxin systems and phage helicase-antibacterial helicase systems. Small proteins that function as inhibitors may be a common strategy for control of cellular processes, thus could inspire the development of new nucleic acid and protein manipulation tools, with diverse biotechnological applications.

microbiology↗

bioRxiv2019

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three new potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14 and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for anti-CRISPR function, and have divergent C-termini that are required in each case for selective inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust and specific inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-15 as unique bifunctional inhibitors of SauCas9.

biochemistry↗

bioRxiv2019

PRC2 activates interferon-stimulated genes indirectly by repressing miRNAs in glioblastoma

Polycomb repressive complex 2 (PRC2) is a chromatin binding complex that represses gene expression by methylating histone H3 at K27 to establish repressed chromatin domains. PRC2 can either regulate genes directly through the methyltransferase activity of its component EZH2 or indirectly by regulating other gene regulators. Gene expression analysis of glioblastoma (GBM) cells lacking EZH2 showed that PRC2 regulates hundreds of interferon-stimulated genes (ISGs). We found that PRC2 directly represses several ISGs and also indirectly activates a distinct set of ISGs. Assessment of EZH2 binding proximal to miRNAs showed that PRC2 directly represses miRNAs encoded in the chromosome 14 imprinted DLK1-DIO3 locus. We found that repression of this locus by PRC2 occurs in immortalized GBM-derived cell lines as well as in primary bulk tumors from GBM and anaplastic astrocytoma patients. Through repression of these miRNAs and several other miRNAs, PRC2 activates a set of ISGs that are targeted by these miRNAs. This PRC2-miRNA-ISG network is likely to be important in regulating gene expression programs in GBM.

molecular biology↗

bioRxiv2018

Identification and removal of sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies

The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Non-specific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published datasets. We show that mispriming can occur with as little as 2 bases of complementarity at the 3 end of the primer followed by intermittent regions of complementarity. We also provide a computational pipeline that identifies cDNA reads produced from RT mispriming, allowing users to filter them out from any aligned dataset. Using this analysis pipeline, we identify thousands of mispriming events in a dozen published datasets from diverse technologies including short RNA-seq, total/mRNA-seq, HITS-CLIP and GRO-seq. We further show how RT-mispriming can lead to misinterpretation of data. In addition to providing a solution to computationally remove RT-misprimed reads, we also propose an experimental solution to avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).

genomics↗

bioRxiv2018

MicroRNAs reinforce repression of PRC2 transcriptional targets independently and through a feed-forward regulatory network with PRC2

Gene expression can be regulated at multiple levels, but it is not known if and how there is broad coordination between regulation at the transcriptional and post-transcriptional levels. Transcription factors and chromatin regulate gene expression transcriptionally, while microRNAs (miRNAs) are small regulatory RNAs that function post-transcriptionally. Systematically identifying the post-transcriptional targets of miRNAs and the mechanism of transcriptional regulation of the same targets can shed light on regulatory networks connecting transcriptional and post-transcriptional control. We used iCLIP (individual crosslinking and immunoprecipitation) for the RISC (RNA-induced silencing complex) component AGO2 and global miRNA depletion to identify genes directly targeted by miRNAs. We found that PRC2 (Polycomb repressive complex 2) and its associated histone mark, H3K27me3, is enriched at hundreds of miRNA-repressed genes. We show that these genes are directly repressed by PRC2 and constitute a significant proportion of direct PRC2 targets. For just over half of the genes co-repressed by PRC2 and miRNAs, PRC2 promotes their miRNA-mediated repression by increasing expression of the miRNAs that are likely to target them. miRNAs also repress the remainder of the PRC2 target genes, but independently of PRC2. Thus, miRNAs post-transcriptionally reinforce silencing of PRC2-repressed genes that are inefficiently repressed at the level of chromatin, by either forming a feed-forward regulatory network with PRC2 or repressing them independently of PRC2.

genomics↗