Delayed mucosal anti-viral responses despite robust peripheral inflammation in fatal COVID-19

Background While inflammatory and immune responses to SARS-CoV-2 infection in peripheral blood are extensively described, responses at the upper respiratory mucosal site of initial infection are relatively poorly defined. We sought to identify mucosal cytokine/chemokine signatures that distinguished COVID-19 severity categories, and relate these to disease progression and peripheral inflammation. Methods We measured 35 cytokines and chemokines in nasal samples from 274 patients hospitalised with COVID-19. Analysis considered the timing of sampling during disease, as either the early (0-5 days post-symptom onset) or late (6-20 days post-symptom onset). Results Patients that survived severe COVID-19 showed IFN-dominated mucosal immune responses (IFN-γ, CXCL10 and CXCL13) early in infection. These early mucosal responses were absent in patients that would progress to fatal disease despite equivalent SARS-CoV-2 viral load. Mucosal inflammation in later disease was dominated by IL-2, IL-10, IFN-γ, and IL-12p70, which scaled with severity but did not differentiate patients who would survive or succumb to disease. Cytokines and chemokines in the mucosa showed distinctions from responses evident in the peripheral blood, particularly during fatal disease. Conclusions Defective early mucosal anti-viral responses anticipate fatal COVID-19 but are not associated with viral load. Early mucosal immune responses may define the trajectory of severe COVID-19

Colorectal Cancer Modeling with Organoids: Discriminating between Oncogenic RAS and BRAF Variants

RAS and BRAF proteins are frequently mutated in colorectal cancer (CRC) and have been associated with therapy resistance in metastatic CRC patients. RAS isoforms are considered to act as redundant entities in physiological and pathological settings. However, there is compelling evidence that mutant variants of RAS and BRAF have different oncogenic potentials and therapeutic outcomes. In this review we describe similarities and differences between various RAS and BRAF oncogenes in CRC development, histology, and therapy resistance. In addition, we discuss the potential of patient-derived tumor organoids for personalized therapy, as well as CRC modeling using genome editing in preclinical model systems to study similarities and discrepancies between the effects of oncogenic MAPK pathway mutations on tumor growth and drug response

Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells

<div><p>The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.</p></div

CRISPR/Cas9 baculovirus mediated Cas9 expression in U-2 OS cells.

<p>A) Schematic representation of the recombination of a pAceBac-Cas9 plasmid with a bacmid in EmBacY cells. The resulting bacmids were used for CRISPR/Cas9 baculovirus production in Sf9 cells. B) Representative FACS-profile showing GFP expression in U-2 OS cells treated with CRISPR/Cas9 baculovirus (MOI: 25). C) Western blot showing expression of Cas9 in U-2 OS cells treated with CRISPR/Cas9 baculoviruses (MOI: 25). α-tubulin was used as a loading control.</p

Effect of CRISPR/Cas9 baculoviral transduction in a panel of cell lines.

<p>A) Representative FACS-profiles showing GFP expression in cells treated with Cas9-GFP baculovirus (MOI: 75). The markers are set such that 2% of the cells treated with Cas9-puro baculovirus are included in this region. The percentage of cells treated with Cas9-GFP baculovirus within the marker region is indicated. B) Immunofluorescence images of mitotic cells treated with the indicated Cas9-GFP baculoviruses (MOI: 75) were scored by eye for the presence or absence of centromeric Aurora B. The bars represent the mean ± SD of 2 experiments. At least 24 cells were analyzed per experiment per condition.</p

Introduction of the H250Y mutation in Aurora B.

<p>A) Schematic representation of the introduction of an HDR template carrying point mutations in the pAceBac-Cas9 plasmid. B) Colony formation of HCT116 cells in ZM447439 with cells carrying either wildtype Aurora B alleles, or alleles with homozygous H250Y mutations, or with a heterozygous mutation for H250Y combined with an indel in the other allele. The numbers indicate colony outgrowth in the ZM447439 treated conditions relative to the untreated conditions. Clones were also tested for their capacity to form colonies in the presence of puromycin. C) Sequence chromatograms of the Aurora B locus in the vicinity of H250. Depicted is the Aurora B sequence trace of wildtype HCT116 cells or clones carrying either the homozygous Aurora B H250Y mutation or the heterozygous Aurora B H250Y mutation combined with an indel. The PAM site is indicated and the arrows point out the base substitutions that were introduced. The corresponding amino acid sequence is shown in the top graph. D) Representative immunofluorescence images of prometaphase cells that have either retained or lost histone H3 Serine 10 (H3S10) phosphorylation after treatment with ZM447439 (0.5 μM). E) Quantification of immunofluorescence images of prometaphase cells depicted in (D) Cells were scored for H3S10ph loss following treatment with ZM447439 (0.5 μM). F) Immunofluorescence images of wildtype HCT116 cells and clones harboring homozygous Aurora B H250Y mutations treated with the indicated CRISPR/Cas9 baculoviruses (MOI: 75) were arrested in mitosis and scored for loss of centromeric Aurora B. G) Representative FACS-profiles showing the DNA content of wildtype HCT116 cells and clones harboring homozygous Aurora B H250Y mutations that were treated with the indicated CRISPR/Cas9 baculoviruses (MOI: 75). Puromycin treatment was used to select for transduced cells and samples were harvested 4 days after transduction.</p

Endogenous tagging of Haspin using CRISPR/Cas9 baculovirus.

<p>A) Schematic representation of the introduction of an HDR template for endogenous tagging of <i>GSG2</i> in the pAceBac-Cas9 plasmid. B) Integration of the YFP-tag at the C-terminus of the gene encoding Haspin was confirmed by PCR using the indicated primer pairs, schematically depicted in Fig 5A. The PCR product obtained using primers 1 and 2 is of the untagged allele, based on its size. C) Representative live cell images of U-2 OS-LacO Haspin-YFP cells and RPE-1 Haspin-YFP cells in prometaphase and interphase. D) Schematic overview depicting the binding of LacI-tagRFP-Pds5B to the LacO repeats. Upon interaction between Pds5B and Haspin an YFP signal can be detected at this ectopic locus. E) Immunofluorescence images of metaphase spreads of U-2 OS-LacO Haspin-YFP cells expressing LacI-tagRFP or LacI-tagRFP-Pds5B and stained for DAPI, YFP and RFP. F) Quantification of Haspin-YFP at the LacO locus. Depicted is the mean of Haspin-YFP normalized over RFP ± SD. Each dot represents a single cell. The data was analyzed using an un-paired Student’s t-test. G) Immunofluorescence images of metaphase spreads of U-2 OS-LacO Haspin-YFP cells expressing LacI-tagRFP or LacI-tagRFP-Pds5B stained for DAPI, H3T3ph and RFP. H) Quantifications of H3T3ph at the LacO locus. Depicted is the mean of H3T3ph levels normalized over RFP ± the SD. Each dot represents a single cell. The data was analyzed using an un-paired Student’s t-test. A minimum of 23 cells was analyzed per experiment.</p

Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling

Ponsioen et al. use a FRET-based ERK biosensor EKAREN5 in patient-derived organoids to show that EGFR activity amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC

Transcriptomic profiles of aging in purified human immune cells

BACKGROUND Transcriptomic studies hold great potential towards understanding the human aging process. Previous transcriptomic studies have identified many genes with age-associated expression levels; however, small samples sizes and mixed cell types often make these results difficult to interpret. - RESULTS Using transcriptomic profiles in CD14+ monocytes from 1,264 participants of the Multi-Ethnic Study of Atherosclerosis (aged 55–94 years), we identified 2,704 genes differentially expressed with chronological age (false discovery rate, FDR ≤ 0.001). We further identified six networks of co-expressed genes that included prominent genes from three pathways: protein synthesis (particularly mitochondrial ribosomal genes), oxidative phosphorylation, and autophagy, with expression patterns suggesting these pathways decline with age. Expression of several chromatin remodeler and transcriptional modifier genes strongly correlated with expression of oxidative phosphorylation and ribosomal protein synthesis genes. 17% of genes with age-associated expression harbored CpG sites whose degree of methylation significantly mediated the relationship between age and gene expression (p &lt; 0.05). Lastly, 15 genes with age-associated expression were also associated (FDR ≤ 0.01) with pulse pressure independent of chronological age. Comparing transcriptomic profiles of CD14+ monocytes to CD4+ T cells from a subset (n = 423) of the population, we identified 30 age-associated (FDR &lt; 0.01) genes in common, while larger sets of differentially expressed genes were unique to either T cells (188 genes) or monocytes (383 genes). At the pathway level, a decline in ribosomal protein synthesis machinery gene expression with age was detectable in both cell types. - CONCLUSIONS An overall decline in expression of ribosomal protein synthesis genes with age was detected in CD14+ monocytes and CD4+ T cells, demonstrating that some patterns of aging are likely shared between different cell types. Our findings also support cell-specific effects of age on gene expression, illustrating the importance of using purified cell samples for future transcriptomic studies. Longitudinal work is required to establish the relationship between identified age-associated genes/pathways and aging-related diseases