Project description:We used a limited proteolysis-coupled mass spectrometry approach to pinpoint binding sites of cortisol on SARS-CoV-2 S1. Binding target identification is based on the principle that small-ligand binding alters (increases or decreases) the protease accessibility of the target protein37,38. The binding of the ligand (cortisol) to the target protein (SARS-CoV-2 S1) induces perturbations/conformational changes at the binding sites which can lead to either facilitating or preventing proteolysis by non-specific proteases (thermolysin and trypsin)37,38. We subjected SARS-CoV-2 S1 incubated with either cortisol or vehicle to proteolysis by thermolysin followed by trypsin digestion and mass spectrometry-based identification of the resultant peptides. We found that cortisol either facilitated or prevented S1 proteolytic cleavage at discrete sites. Six unique peptides were identified that were only present for SARS-CoV-2 S1 incubated with either cortisol or vehicle.
Project description:Human nontransformed retinal pigment epithelia RPE-PPM1D-T2 cells carrying a truncating mutation in exon 6 of the PPM1D were exposed to ionising radiation (3 Gy) and subsequently were grown in semisolid media for 8 weeks. Six spheroid clones (RPE-PPM1D-T2-SA clones 1-6) were recovered and then were cultivated in adherent conditions. RNA was isolated from asynchronically growing parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA-1 to 6 cells and was subjected to whole exome sequencing. RNA sequencing libraries were prepared using KAPA RNA HyperPrep Kit (Roche) and were sequenced on the NovaSeq 6000 system using NovaSeq S1 Reagent Kit v1.5, 200 cycles (Illumina) with mean coverage >120 for RNA samples, respectively. RNA fastq files were mapped to the hg19 reference using Novoalign (novoalign_2.08.03). PCR duplicates were removed from the BAM files using Picard Tools (picard-tools 1.129), and variant calling was performed using GATK HaplotypeCaller (3.8). RNA fastq files were mapped to the hg19 reference using STAR (STAR-2.5.2b). The PCR duplicates were removed using Picard Tools (picard-tools 1.129). All parts of RNAseq data analysis were conducted in R, version 4.3.2. and RNAseq read counts were normalized using R package DESeq2. Fold change (FC) and log2FC were calculated from normalized reads, nontransformed RPE-PPM1D-T1 cells were considered a reference. Significance of differential expression for each gene was evaluated by Fisher's t-test with simulated p-values and Holm's p-value correction for multiple comparisons.
Project description:To search for host factors regulating SARS-COV-2 infection, we performed a genome-wide loss-of-function CRISPR/Cas9 screen in haploid human ESCs. The regulators were identified by the quantification of enrichment of their mutant clones within a pooled loss-of-function library upon SARS-COV-2 infection.
Project description:To investigate the internal regulatory mechanisms governing the CD28 gene region during T cell activation and to screen for truly active dynamic chromatin regulatory elements responsive to stimulation, we employed the CRISPRa gene editing technology to design specific sgRNAs targeting this region and conducted a tilling CRISPRa screen sequencing on the CD28 gene region.
Project description:Otx2 has been shown to be non cell autonomously required for photoreceptor cell survival in the adult mouse RPE. This study aims to identify Otx2 DNA binding profile in both RPE and neural retina to i) identify direct targets of Otx2 in the RPE ii) compare Otx2 binding profile in neural retina and RPE to unveil hidden functions in the neural retina. WT and GFP antibodies were used to perform two independent ChIP-seq experiments using Illumina GAIIx.
Project description:SARS-CoV-2 has spread globally and caused the COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 are in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected mice. Whereas Fc effector functions are fully dispensable when mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact but not LALA-PG loss of Fc effector function variant mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and enhanced tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Our study demonstrates that therapeutic neutralizing mAbs require Fc effector functions to reduce SARS-CoV-2 infection and modulate protective immune responses.
Project description:Human nontransformed retinal pigment epithelia RPE-PPM1D-T2 cells carrying a truncating mutation in exon 6 of the PPM1D were exposed to ionising radiation (3 Gy) and subsequently were grown in semisolid media for 8 weeks. Six spheroid clones (RPE-PPM1D-T2-SA clones 1-6) were recovered and then were cultivated in adherent conditions. DNA was isolated from asynchronically growing parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA-1 to 6 cells and was subjected to whole exome sequencing. DNA sequencing libraries were prepared using KAPA EvoPlus Kit (Roche) and were sequenced on the NovaSeq 6000 system using NovaSeq S1 Reagent Kit v1.5, 200 cycles (Illumina) with mean coverage >35 DNA samples, respectively. DNA fastq files were mapped to the hg19 reference using Novoalign (novoalign_2.08.03). PCR duplicates were removed from the BAM files using Picard Tools (picard-tools 1.129), and variant calling was performed using GATK HaplotypeCaller (3.8). Copy number variations (CNV) were analyzed using CNVkit version 0.7.4. Areas with median coverage >20 were included in the analysis. RNA fastq files were mapped to the hg19 reference using STAR (STAR-2.5.2b). The PCR duplicates were removed using Picard Tools (picard-tools 1.129).