Reconstituting regulation of the canonical Wnt pathway by engineering a minimal β-catenin destruction machine

Negatively regulating key signaling pathways is critical to development and altered in cancer. Wnt signaling is kept off by the destruction complex, which is assembled around the tumor suppressors APC and Axin and targets β-catenin for destruction. Axin and APC are large proteins with many domains and motifs that bind other partners. We hypothesized that if we identified the essential regions required for APC:Axin cooperative function and used these data to design a minimal β-catenin-destruction machine, we would gain new insights into the core mechanisms of destruction complex function. We identified five key domains/motifs in APC or Axin that are essential for their function in reconstituting Wnt regulation. Strikingly, however, certain APC and Axin mutants that are nonfunctional on their own can complement one another in reducing β-catenin, revealing that the APC:Axin complex is a highly robust machine. We used these insights to design a minimal β-catenin-destruction machine, revealing that a minimized chimeric protein covalently linking the five essential regions of APC and Axin reconstitutes destruction complex internal structure, size, and dynamics, restoring efficient β-catenin destruction in colorectal tumor cells. On the basis of our data, we propose a new model of the mechanistic function of the destruction complex as an integrated machine

Brief Report: Deficiency of Complement 1r Subcomponent in Early-Onset Systemic Lupus Erythematosus: The Role of Disease-Modifying Alleles in a Monogenic Disease

Objective: To identify a genetic cause of early-onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease. Methods: We performed whole-exome sequencing and single-nucleotide polymorphism array genotyping in family members with and without SLE. Protein and gene expression, cytokine profile, neutrophil extracellular trap (NET) formation, and presence of low-density granulocytes were evaluated in patient primary cells and serum samples. Results: We identified a novel, homozygous, loss-of-function mutation (p.Pro445Leufs*11) in the C1R gene. Using the Sanger method of DNA sequencing in 14 family members, we confirmed the presence of the mutation in 4 patients with SLE and in an asymptomatic 9-year-old girl. Complement levels were low in sera from patients with truncated C1r protein. Two siblings with SLE who were available for detailed evaluation exhibited strong type I interferon (IFN) inflammatory signatures despite their disease being clinically inactive at the time of sampling. The type I IFN transcriptional signature in the patients’ blood correlated with disease expressivity, whereas the neutrophil signature in peripheral blood mononuclear cells was likely associated with disease severity. The female patient with SLE with the most severe phenotype presented with a stronger neutrophil signature, defined by enhanced NET formation and the presence of low-density granulocytes. Analysis of exome data for modifying alleles suggested enrichment of common SLE-associated variants in the more severely affected patients. Lupus-associated HLA alleles or HLA haplotypes were not shared among the 4 affected subjects. Conclusion: Our findings revealed a novel high-penetrance mutation in C1R as the cause of monogenic SLE. Disease expressivity in this family appears to be influenced by additional common and rare genetic variants

Evaluation and Management of Deficiency of Adenosine Deaminase 2: An International Consensus Statement

IMPORTANCE: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited disease characterized by systemic vasculitis, early-onset stroke, bone marrow failure, and/or immunodeficiency affecting both children and adults. DADA2 is among the more common monogenic autoinflammatory diseases, with an estimate of more than 35 000 cases worldwide, but currently, there are no guidelines for diagnostic evaluation or management. OBJECTIVE: To review the available evidence and develop multidisciplinary consensus statements for the evaluation and management of DADA2. EVIDENCE REVIEW: The DADA2 Consensus Committee developed research questions based on data collected from the International Meetings on DADA2 organized by the DADA2 Foundation in 2016, 2018, and 2020. A comprehensive literature review was performed for articles published prior to 2022. Thirty-two consensus statements were generated using a modified Delphi process, and evidence was graded using the Oxford Center for Evidence-Based Medicine Levels of Evidence. FINDINGS: The DADA2 Consensus Committee, comprising 3 patient representatives and 35 international experts from 18 countries, developed consensus statements for (1) diagnostic testing, (2) screening, (3) clinical and laboratory evaluation, and (4) management of DADA2 based on disease phenotype. Additional consensus statements related to the evaluation and treatment of individuals with DADA2 who are presymptomatic and carriers were generated. Areas with insufficient evidence were identified, and questions for future research were outlined. CONCLUSIONS AND RELEVANCE: DADA2 is a potentially fatal disease that requires early diagnosis and treatment. By summarizing key evidence and expert opinions, these consensus statements provide a framework to facilitate diagnostic evaluation and management of DADA2

By Bread Alone: A Short Guide to Celiac Disease, Gluten, and Life

My journey with CD has been relatively easy, because I happened to be ensconced in a community that supported me, and I was given the tools I needed to adhere to my diet. My hope for this piece is that it will help to elucidate some the complexities of a food-based illness and demonstrate a few ways of achieving some of the support that I gained from my community.Bachelor of Art

Revisiting TNF Receptor-Associated Periodic Syndrome (TRAPS): Current Perspectives

Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal dominant autoinflammatory syndrome characterized by prolonged and recurrent episodes of fever, abdominal and/or chest pain, arthralgia, myalgia, and erythematous rash. TRAPS is associated with heterozygous variants in the TNFRSF1A gene, which encodes the TNFR1 (tumor necrosis factor receptor 1) receptor. Disease-causing variants are found exclusively in the extracellular domain of TNFR1 and affect receptor structure and binding to the TNF ligand. The precise mechanism of the disease is still unclear, but it is thought that intracellular accumulation of misfolded mutant protein leads to endoplasmic reticulum stress and enhanced inflammatory responses through constitutive activation of various immune pathways. Other possible mechanisms contributing to the disease pathogenesis include defective receptor shedding, TNF-induced cell death, production of reactive oxygen species, and autophagy impairment. Patients’ leucocytes are hyperresponsive to stimulation and produce elevated levels of proinflammatory cytokines. Systemic autoimmune (AA) amyloidosis is an important cause of morbidity and mortality in TRAPS. Over the last two decades, new therapies have changed the progression and outcome of the disease. In this review, we summarize clinical data from 209 patients with validated pathogenic variants reported in the literature and discuss TRAPS diagnosis, pathogenesis, and treatment options

Miraprepped plasmids can be effectively used to transfect human tissue culture cells.

<p>(A) Immunofluorescence of human SW480 cells transfected with a plasmid encoding GFP (3 kb; Miraprepped using 1x volume of ethanol+50 μg/ml RNase) and stained for β-catenin via antibody. SW480 cells have high levels of the Wnt transcriptional co-activator β-catenin due to a mutation in one of its key negative regulators, APC. (A’) GFP is uniformly distributed throughout transfected cells. (A”) Expression of GFP does not alter β-catenin levels—arrows compare a transfected and an untransfected cell. (B) Immunofluorescence of SW480 cells transfected with a plasmid encoding GFP-tagged <i>Drosophila</i> APC2 (8 kb; Miraprep using 1x volume of ethanol+50 μg/ml RNase). (B’) APC2 is uniformly distributed in the cytoplasm. (B”) Fly APC2 is able to reduce β-catenin levels, thus compensating for the mutation of the endogenous human APC in the SW480 cells. (C) Transfection efficiency into SW480 cells is similar for Miraprepped samples (using 1x volume of ethanol+50 μg/ml RNase) and those transfected with DNA prepared via the standard Qiagen Maxiprep procedure. 2 μg of plasmids encoding GFP (3 kb) or GFP-tagged Drosophila APC2 (8 kb) were transfected using Lipofectamine 2000. 100 cells were counted in each of three independent experiments. (D) Immunoblot analysis of transfection efficiency. 3 conditions were tested: DNAs prepared by Miniprep (GeneJET), Miraprep (using 1x volume ethanol), and Miraprep (using 1x vol+50 μg/ml RNase). All led to roughly equal levels of protein expression. Lipofectamine 2000 and 2 μg of plasmid DNA were used. Cells were directly lysed in SDS-loading buffer. aPKCγ was used as the loading control.</p

The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields

<div><p>Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs.</p></div

Different RNase concentrations do not reduce DNA yield in Miraprepped samples and Miraprep is not significantly contaminated by low molecular weight RNA.

<p>(A) Standard Miniprep, or Miraprepped plasmids prepared using 1x volume of ethanol, were treated with indicated RNase concentration, added freshly into the resuspension buffer before beginning the procedure. Top: 0.4 μg was electrophoresed on an agarose gel. DNA concentration only varied slightly when RNase was freshly added. Bottom: OD260/280 ratio. (B,C) Testing for low molecular weight RNA in Miniprep and Miraprep samples, respectively. (B) Miraprep and Miniprep samples of the 8 kb plasmid contain little or no small molecular weight RNA. Pre-column = after alkaline lysis, Flow-through = flow-through of spin column, Final lane in each set is eluted plasmid. 10 μl of pre-column and flow-through samples were loaded, while 2 μl were loaded of Miniprep or Miraprep samples. (C) Miniprep and Miraprep samples of the 14 kb plasmid have little to no low molecular RNA present. Loading same as described in (B).</p

Comparison of commercial plasmid preparation methods from three manufacturer’s (GeneJet, Qiagen, and GenElute) with the Miracle-prep.

<p>Comparison of commercial plasmid preparation methods from three manufacturer’s (GeneJet, Qiagen, and GenElute) with the Miracle-prep.</p

Testing whether the columns might act as a filter and verifying DNA yields using comparison to known DNA standards.

<p>(A) To determine whether silica spin columns might capture DNA by acting as filters, the Miraprep procedure was followed through the neutralization step with 1x volume of ethanol added or no ethanol added as a control, and then the sample was passed over a simple centrifugal filter (pore size 0.22 μm), the filter was washed following the Miraprep protocol, DNA was eluted from the top surface of the filter, and electrophoresed on an agarose gel. DNA was only recovered after ethanol addition. (B) Silica columns are more efficient than centrifugal filters in capturing plasmid DNA. Comparison of DNA yields using silica columns or centrifugal filter columns; from three independently conducted Mirapreps. (C,D) Standard Miniprep plasmids, or Miraprep plasmids prepared using our final protocol using 1x volume of ethanol, were electrophoresed on an agarose gel and amounts compared to known DNA standards (Thermo Scientific GeneRuler 1 kb DNA Ladder #SM0312 (0.5 μg/μl)). 2 μl DNA plus 5 μl loading buffer were loaded in each lane. (C) GenElute kit. (D) GeneJET kit. Above each gel is the DNA amount calculated from OD260 and below the gel estimates from comparison to DNA markers of known amounts. Image J was used to quantify DNA band intensities in (C,D). DNA amounts calculated by both methods were comparable.</p