The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation.

Activation of RAS is crucial in driving cellular outcomes including proliferation, differentiation, migration and apoptosis via the MAPK pathway. This is initiated on recruitment of Grb2, as part of a Grb2-Sos complex, to an up-regulated receptor tyrosine kinase (RTK), enabling subsequent interaction of Sos with the plasma membrane-localised RAS. Aberrant regulation at this convergence point for RTKs in MAPK signalling is a key driver of multiple cancers. Splicing of the GRB2 gene produces a deletion variant, Grb3-3, that is incapable of binding to RTKs. We show that, despite maintaining the ability to bind to Sos, the Grb3-3-Sos complex remains in the cytoplasm, unable to engage with RAS. Competition between Grb2 and Grb3-3 for binding to C-terminal proline-rich sequences on Sos modulates MAPK signalling. Additionally, we demonstrate that splicing is regulated by heterogenous nuclear riboproteins C1/C2, and that normal and malignant colon tissue show differential Grb3-3 expression.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Risk perception and knowledge of COVID-19 in patients with celiac disease.

BACKGROUND: We recently demonstrated that the odds of contracting coronavirus disease 2019 (COVID-19) in patients with celiac disease (CeD) is similar to that of the general population. However, how patients with CeD perceive their COVID-19 risk may differ from their actual risk. AIM: To investigate risk perceptions of contracting COVID-19 in patients with CeD and determine the factors that may influence their perception. METHODS: We distributed a survey throughout 10 countries between March and June 2020 and collected data on demographics, diet, COVID-19 testing, and risk perceptions of COVID-19 in patients with CeD. Participants were recruited through various celiac associations, clinic visits, and social media. Risk perception was assessed by asking individuals whether they believe patients with CeD are at an increased risk of contracting COVID-19 when compared to the general population. Logistic regression was used to determine the influencing factors associated with COVID-19 risk perception, such as age, sex, adherence to a gluten-free diet (GFD), and comorbidities such as cardiac conditions, respiratory conditions, and diabetes. Data was presented as adjusted odds ratios (aORs). RESULTS: A total of 10737 participants with CeD completed the survey. From them, 6019 (56.1%) patients with CeD perceived they were at a higher risk or were unsure if they were at a higher risk of contracting COVID-19 compared to the non-CeD population. A greater proportion of patients with CeD perceived an increased risk of contracting COVID-19 when compared to infections in general due to their CeD (56.1% vs 26.7%, P < 0.0001). Consequently, 34.8% reported taking extra COVID-19 precautions as a result of their CeD. Members of celiac associations were less likely to perceive an increased risk of COVID-19 when compared to non-members (49.5% vs 57.4%, P < 0.0001). Older age (aOR: 0.99; 95%CI: 0.99 to 0.99, P < 0.001), male sex (aOR: 0.84; 95%CI: 0.76 to 0.93, P = 0.001), and strict adherence to a GFD (aOR: 0.89; 95%CI: 0.82 to 0.96, P = 0.007) were associated with a lower perception of COVID-19 risk and the presence of comorbidities was associated with a higher perception of COVID-19 risk (aOR: 1.38; 95%CI: 1.22 to 1.54, P < 0.001). CONCLUSION: Overall, high levels of risk perceptions, such as those found in patients with CeD, may increase an individual's pandemic-related stress and contribute to negative mental health consequences. Therefore, it is encouraged that public health officials maintain consistent communication with the public and healthcare providers with the celiac community. Future studies specifically evaluating mental health in CeD could help determine the consequences of increased risk perceptions in this population.

The Risk of Contracting COVID-19 Is Not Increased in Patients With Celiac Disease.

The World Health Organization declared coronavirus disease-2019 (COVID-19) a global pandemic in March 2020. Since then, there are more than 34 million cases of COVID-19 leading to more than 1 million deaths worldwide. Numerous studies suggest that celiac disease (CeD), a chronic immune-mediated gastrointestinal condition triggered by gluten, is associated with an increased risk of respiratory infections.1-3 However, how it relates to the risk of COVID-19 is unknown. To address this gap, we conducted a cross-sectional study to evaluate whether patients with self-reported CeD are at an increased risk of contracting COVID-19.

Probiotics for Celiac Disease: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

INTRODUCTION: Many patients with celiac disease (CD) experience persistent symptoms despite adhering to the gluten-free diet. Different studies have assessed the use of probiotics as an adjuvant treatment for CD. We performed a systematic review and meta-analysis to evaluate the efficacy of probiotics in improving gastrointestinal (GI) symptoms and quality of life (QOL) in patients with CD. METHODS: We searched EMBASE, MEDLINE, CINAHL, Web of Science, CENTRAL, and DARE databases up to February 2019 for randomized controlled trials (RCTs) evaluating probiotics compared with placebo for treating CD. We collected data on GI symptoms, QOL, adverse events, serum tumor necrosis factor-α, intestinal permeability, and microbiota composition. RESULTS: We screened 2,831 records and found that 7 articles describing 6 RCTs (n = 279 participants) were eligible for quantitative analysis. Probiotics improved GI symptoms when assessed by the GI Symptoms Rating Scale (mean difference symptom reduction: -28.7%; 95% confidence interval [CI] -43.96 to -13.52; P = 0.0002). There was no difference in GI symptoms after probiotics when different questionnaires were pooled. The levels of Bifidobacteria increased after probiotics (mean difference: 0.85 log colony-forming units (CFU) per gram; 95% CI 0.38-1.32 log CFU per gram; P = 0.0003). There were insufficient data on tumor necrosis factor-a levels or QOL for probiotics compared with placebo. No difference in adverse events was observed between probiotics and placebo. The overall certainty of the evidence ranged from very low to low. DISCUSSION: Probiotics may improve GI symptoms in patients with CD. High-quality clinical trials are needed to improve the certainty in the evidence (see Visual abstract, Supplementary Digital Content 2, http://links.lww.com/AJG/B595).

Association Between Inflammatory Bowel Diseases and Celiac Disease: A Systematic Review and Meta-Analysis.

BACKGROUND & AIMS: There is controversy over the association between celiac disease (CeD) and inflammatory bowel diseases (IBD). We performed a systematic review and meta-analysis to assess evidence for an association between CeD and IBD. METHODS: We searched databases including MEDLINE, EMBASE, CENTRAL, Web of Science, CINAHL, DARE, and SIGLE through June 25, 2019 for studies assessing the risk of CeD in patients with IBD, and IBD in patients with CeD, compared with controls of any type. We used the Newcastle-Ottawa Scale to evaluate the risk of bias and GRADE to assess the certainty of the evidence. RESULTS: We identified 9791 studies and included 65 studies in our analysis. Moderate certainty evidence found an increased risk of CeD in patients with IBD vs controls (risk ratio [RR] 3.96; 95% confidence interval [CI] 2.23-7.02) and increased risk of IBD in patients with CeD vs controls (RR 9.88; 95% CI 4.03-24.21). There was low-certainty evidence for the risk of anti-Saccharomyces antibodies, a serologic marker of IBD, in patients with CeD vs controls (RR 6.22; 95% CI 2.44-15.84). There was low-certainty evidence for no difference in risk of HLA-DQ2 or DQ8 in patients with IBD vs controls (RR 1.04; 95% CI 0.42-2.56), and very low-certainty evidence for an increased risk of anti-tissue transglutaminase in patients with IBD vs controls (RR 1.52; 95% CI 0.52-4.40). Patients with IBD had a slight decrease in risk of anti-endomysial antibodies vs controls (RR 0.70; 95% CI 0.18-2.74), but these results are uncertain. CONCLUSIONS: In a systematic review and meta-analysis, we found an increased risk of IBD in patients with CeD and increased risk of CeD in patients with IBD, compared with other patient populations. High-quality prospective cohort studies are needed to assess the risk of CeD-specific and IBD-specific biomarkers in patients with IBD and CeD.

Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

BACKGROUND: Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). METHODS: Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. RESULTS: Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2-14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. CONCLUSIONS: Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material.

Histone deacetylase 3 indirectly modulates tubulin acetylation.

Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3-silencing mediator of retinoic and thyroid receptors (SMRT)-deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation.

Microfluidic studies of CO2 sequestration by frustrated Lewis pairs.

Frustrated Lewis pairs (FLPs) comprising sterically hindered Lewis acids and bases offer the capability to reversibly capture CO2 under mild reaction conditions. The determination of equilibrium constants and thermodynamic properties of these reactions should enable assessment of the efficiency of a particular FLP system for CO2 sequestration and provide insights for design of new, efficient formulations of FLP catalysts for CO2 capture. We have developed a microfluidic approach to studies of FLP-CO2 reactions, which provides their thermodynamic characterization that is not accessible otherwise. The approach enables the determination of the equilibrium reaction constants at different temperatures, the enthalpy, the entropy, and the Gibbs energy of these reactions, as well as the enhancement factor. The microfluidic methodology has been validated by applying it to the well-characterized reaction of CO2 with a secondary amine. The microfluidic approach can be applied for fundamental thermodynamic studies of other gas-liquid reactions.