Genome-wide CRISPR screening identifies new regulators of glycoprotein secretion.

Background: The fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. Methods: With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen, followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. Results: We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5. Conclusions: This study has identified new factors for protein secretion involved in Golgi homeostasis

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response, but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)- 6. These late immunometabolic and immune defects may have clinical implication

Research data supporting 'Genome-wide CRISPR screening identifies new regulators of glycoprotein secretion'

Files S1 - S6 contain underlying data for figures published in the paper. S1: sgRNA counts of unsorted and sorted population from the CRISPR screen detailed in the paper, which aimed to identify genes that regulate glycoprotein secretion. Sheet 1 is a table of sgRNA counts, including the gene name that each sgRNA targets. Sheet 2 includes full descriptions of the populations. S2: MaGECK (version 0.5.7) analysis of sgRNA counts from the CRISPR screen. Both MaGECK-MLE and MaGECK-RRA results are shown. False discovery rates (FDR) from this table were used to create figure 1C-E S3: Raw FCS files from flow cytometry staining of sorted samples, used to create figure 1B. S4: Raw chemiluminescence data, used to create figure 2. S5: Unedited image files used in Figures 3D, 4 and 5. S6: Data output from Cell Profiler, showing total number of cells identified per image and number of cells classified as having fragmented or intact Golgi. E1-E4 is extended data for figures and tables published in the paper. E1: Figure of all genes analysed for having fragmented Golgi; top hits from this talbe are shown in Figure 3C. Percentage of cells with fragmented Golgi for all of the hits screened in the tertiary screen. As in Figure 3C, hits are arranged alphabetically and coloured by cell count, with darker blue spots representing more confluent wells. E2: Sequence of P5 primers used for PCR amplification of sgRNA. E3: Sequence of P7 primers used for PCR amplification of sgRNA. E4: Details of the siGenome pooled siRNA library used in secondary screening

Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue.

At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips

Vascularisation (von Willebrand factor expression) in cryopreserved medulla-containing ovarian tissue after in vitro and CAM-culture.

<p>Immonostained after in vitro (a, A) and CAM (b, B) culture. Bar = 0.25 mm.</p