The Wnt secretion protein Evi/Gpr177 promotes glioma tumourigenesis

Malignant astrocytomas are highly aggressive brain tumours with poor prognosis. While a number of structural genomic changes and dysregulation of signalling pathways in gliomas have been described, the identification of biomarkers and druggable targets remains an important task for novel diagnostic and therapeutic approaches. Here, we show that the Wnt-specific secretory protein Evi (also known as GPR177/Wntless/Sprinter) is overexpressed in astrocytic gliomas. Evi/Wls is a core Wnt signalling component and a specific regulator of pan-Wnt protein secretion, affecting both canonical and non-canonical signalling. We demonstrate that its depletion in glioma and glioma-derived stem-like cells led to decreased cell proliferation and apoptosis. Furthermore, Evi/Wls silencing in glioma cells reduced cell migration and the capacity to form tumours in vivo. We further show that Evi/Wls overexpression is sufficient to promote downstream Wnt signalling. Taken together, our study identifies Evi/Wls as an essential regulator of glioma tumourigenesis, identifying a pathway-specific protein trafficking factor as an oncogene and offering novel therapeutic options to interfere with the aberrant regulation of growth factors at the site of production

Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells

Aberrant regulation of the Wnt/β-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or β-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or β-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind β-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or β-catenin depend on Wnt ligands and their secretion for a sufficient level of β-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Undenatured parvovirus B19 antigens are essential for the accurate detection of parvovirus B19 IgG

Recombinant versions of parvovirus B19 capsid proteins VP1 and VP2 are used for immunodiagnostic assays for detection of antiviral antibodies. The immune response to B19 is characterized by a gradual loss of antibodies directed against linear epitopes of VP2. A similar occurrence for antibodies raised against VP1 protein would represent a limitation to serological assays incorporating denatured versions of either viral antigen. Four detection systems for B19 Ig detection have been developed, including an IgG enzyme immunoassay (EIA) based on undenatured VP2, an immunofluorescence assay (IFA) based on undenatured VP1, a Western blot assay incorporating denatured VP1 and VP2, and an alternative blot system using denatured VP1 but undenatured VP2. Specimens (n=108) were tested by all four systems and identical results were obtained by EIA, IFA, and alternative blot systems, whereby 75/108 (69%) were B19 IgG-positive. Twelve B19 IgG-positive specimens, representing 16% (12/75) of the confirmed positives, did not react to either viral antigens when tested by Western blot. It is concluded that these sera do not react with linear epitopes of VP1 and VP2 antigens. Eighty-five different specimens, which had previously been shown to be both B19 IgM- and IgG-positive by EIA and IFA, were positive by B19 IgM and IgG Western blot. In the IgG Western blot assay, 69 reacted with both VP1 and VP2 and 16 with VP1 only. It is concluded that there is a requirement for at least one undenatured antigen for the immunological detection of B19 IgG

EDGE-WEIGHTING OF GENE EXPRESSION GRAPHS

In recent years, considerable research efforts have been directed to micro-array technologies and their role in providing simultaneous information on expression profiles for thousands of genes. These data, when subjected to clustering and classification procedures, can assist in identifying patterns and providing insight on biological processes. To understand the properties of complex gene expression datasets, graphical representations can be used. Intuitively, the data can be represented in terms of a bipartite graph, with weighted edges corresponding to gene-sample node couples in the dataset. Biologically meaningful subgraphs can be sought, but performance can be influenced both by the search algorithm, and, by the graph-weighting scheme and both merit rigorous investigation. In this paper, we focus on edge-weighting schemes for bipartite graphical representation of gene expression. Two novel methods are presented: the first is based on empirical evidence; the second on a geometric distribution. The schemes are compared for several real datasets, assessing efficiency of performance based on four essential properties: robustness to noise and missing values, discrimination, parameter influence on scheme efficiency and reusability. Recommendations and limitations are briefly discussed.Edge-weighting, weighted graphs, gene expression, bi-clustering

Detection of parvovirus B19 IgM by antibody capture enzyme immunoassay: receiver operating characteristic analysis

Parvovirus B19 infection can cause severe effects in high-risk groups including pregnant women and immunocompromised individuals. Although serological detection of B19 infection is commonplace, minimal information is available on the absolute performance characteristics of various tests for the detection of B19 IgM. The performance of the first parvovirus B19 IgM enzyme immunoassay to be cleared by the US Food and Drug Administration (FDA) is described. The immunoassay cut-off has been established using receiver operating characteristic (ROC) analysis giving a sensitivity and specificity of detection of 89.1 and 99.4%, respectively. No cross-reactivity is observed with rubella or other viral disease IgM which cause similar symptomologies to parvovirus B19. Multi-site reproducibility studies have shown high immunoassay reproducibility with detection rates (observed:expected result) of 100% for nonreactive specimens (N=324) and strongly reactive (N=403), respectively. Immunoassay reproducibility ranged from 11.76 to 17.46% coefficient of variation for all reactive specimens tested (N=12) whereby each specimen was assayed a total of 81 times. Parvovirus B19 IgM seroprevalence of 1% was observed in a US blood donor population (N=399). In the absence of international performance criteria, this study will be of major benefit to the clinical virologist in assessing immunoassay reliability for the detection of recent infection with parvovirus B19

Degron mediated BRM/SMARCA2 depletion uncovers novel combination partners for treatment of BRG1/SMARCA4-mutant cancers.

Recent studies have highlighted that cancer cells with a loss of the SWI/SNF complex catalytic subunit BRG1 are dependent on the remaining ATPase, BRM, making it an attractive target for cancer therapy. However, an understanding of the extent of target inhibition required to arrest cell growth, necessary to develop an appropriate therapeutic strategy, remains unknown. Here, we utilize tunable depletion of endogenous BRM using the SMASh degron, and interestingly observe that BRG1-mutant lung cancer cells require near complete depletion of BRM to robustly inhibit growth both in vitro and in vivo. Therefore, to identify pathways that synergize with partial BRM depletion and afford a deeper response, we performed a genome-wide CRISPR screen and discovered a combinatorial effect between BRM depletion and the knockout of various genes of the oxidative phosphorylation pathway and the anti-apoptotic gene MCL1. Together these studies provide an important framework to elucidate the requirements of BRM inhibition in the BRG1-mutant state with implications on the feasibility of targeting BRM alone, as well as reveal novel insights into pathways that can be exploited in combination toward deeper anti-tumor responses

The impact of covid-19 on psychosocial well-being and learning for australian nursing and midwifery undergraduate students: a cross-sectional survey

Aim: To explore the impact of COVID-19 on psychosocial well-being and learning for nursing and midwifery undergraduate students in an Australian university. Background: The World Health Organization has reported a substantial psychological impact of COVID-19 on healthcare professionals to date. Evidence is lacking, however, regarding university nursing and midwifery students of the pandemic and its impact on their educational preparation and/or clinical placement during the COVID-19 pandemic. Design: Cross-sectional survey of nursing and midwifery undergraduate students enrolled in the Bachelor of Nursing suite of courses from the study institution in August- September 2020. Methods: A cross-sectional self-administered anonymous online survey was distributed to current nursing and midwifery undergraduate students. The survey included three open-ended questions; responses were thematically analysed. Results: Of 2907 students invited, 637 (22%) responded with 288 of the respondents (45%) providing a response to at least one of the three open-ended questions. Three major themes associated with the impact of the pandemic on psychosocial well-being and learning were identified: psychosocial impact of the pandemic, adjustment to new modes of teaching and learning, and concerns about course progression and career. These themes were underpinned by lack of motivation to study, feeling isolated, and experiencing stress and anxiety that impacted on students’ well-being and their ability to learn and study. Conclusions: Students were appreciative of different and flexible teaching modes that allowed them to balance their study, family, and employment responsibilities. Support from academic staff and clinical facilitators/mentors combined with clear and timely communication of risk management related to personal protective equipment (PPE) in a healthcare facility, were reported to reduce students’ stress and anxiety. Ways to support and maintain motivation among undergraduate nursing and midwifery students are needed. © 202

Amplicon sequencing of colorectal cancer: variant calling in frozen and formalin-fixed samples.

Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications