Genetic and epigenetic profiling of BRCA1/2 in ovarian tumors reveals additive diagnostic yield and evidence of a genomic BRCA1/2 DNA methylation signature

Poly-ADP-ribose-polymerase inhibitor (PARPi) treatment is indicated for advanced-stage ovarian tumors with BRCA1/2 deficiency. The “BRCAness” status is thought to be attributed to a tumor phenotype associated with a specific epigenomic DNA methylation profile. Here, we examined the diagnostic impact of combined BRCA1/2 sequence, copy number, and promoter DNA methylation analysis, and evaluated whether genomic DNA methylation patterns can predict the BRCAness in ovarian tumors. DNA sequencing of 172 human tissue samples of advanced-stage ovarian adenocarcinoma identified 36 samples with a clinically significant tier 1/2 sequence variants (point mutations and in/dels) and 9 samples with a CNV causing a loss of function in BRCA1/2. DNA methylation analysis of the promoter of BRCA1/2 identified promoter hypermethylation of BRCA1 in two mutation-negative samples. Computational modeling of genome-wide methylation markers, measured using Infinium EPIC arrays, resulted in a total accuracy of 0.75, sensitivity: 0.83, specificity: 0.64, positive predictive value: 0.76, negative predictive value: 0.74, and area under the receiver’s operating curve (AUC): 0.77, in classifying tumors harboring a BRCA1/2 defect from the rest. These findings indicate that the assessment of CNV and promoter DNA methylation in BRCA1/2 increases the cumulative diagnostic yield by 10%, compared with the 20% yield achieved by sequence variant analysis alone. Genomic DNA methylation data can partially predict BRCAness in ovarian tumors; however, further investigation in expanded BRCA1/2 cohorts is needed, and the effect of other double strand DNA repair gene defects in these tumors warrants further investigations

Gene coexpression clusters and putative regulatory elements underlying seed storage reserve accumulation in Arabidopsis

Abstract Background In Arabidopsis, a large number of genes involved in the accumulation of seed storage reserves during seed development have been characterized, but the relationship of gene expression and regulation underlying this physiological process remains poorly understood. A more holistic view of this molecular interplay will help in the further study of the regulatory mechanisms controlling seed storage compound accumulation. Results We identified gene coexpression networks in the transcriptome of developing Arabidopsis (Arabidopsis thaliana) seeds from the globular to mature embryo stages by analyzing publicly accessible microarray datasets. Genes encoding the known enzymes in the fatty acid biosynthesis pathway were found in one coexpression subnetwork (or cluster), while genes encoding oleosins and seed storage proteins were identified in another subnetwork with a distinct expression profile. In the triacylglycerol assembly pathway, only the genes encoding diacylglycerol acyltransferase 1 (DGAT1) and a putative cytosolic "type 3" DGAT exhibited a similar expression pattern with genes encoding oleosins. We also detected a large number of putative cis-acting regulatory elements in the promoter regions of these genes, and promoter motifs for LEC1 (LEAFY COTYLEDON 1), DOF (DNA-binding-with-One-Finger), GATA, and MYB transcription factors (TF), as well as SORLIP5 (Sequences Over-Represented in Light-Induced Promoters 5), are overrepresented in the promoter regions of fatty acid biosynthetic genes. The conserved CCAAT motifs for B3-domain TFs and binding sites for bZIP (basic-leucine zipper) TFs are enriched in the promoters of genes encoding oleosins and seed storage proteins. Conclusions Genes involved in the accumulation of seed storage reserves are expressed in distinct patterns and regulated by different TFs. The gene coexpression clusters and putative regulatory elements presented here provide a useful resource for further experimental characterization of protein interactions and regulatory networks in this process.</p

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

Insulin increases GLUT4 at the muscle cell surface, and this process requires actin remodeling. We show that a dynamic cycle of actin polymerization and severing is induced by insulin, governed by Arp2/3 and dephosphorylation of cofilin, respectively. The cycle is self-perpetuating and is essential for GLUT4 translocation

PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis

Application of robust estimation in proficiency testing of laboratory by low number of measurements

W pracy przedstawiono zalety odpornej iteracyjnej metody szacowania wskaźników dokładności pomiarów dla oceny biegłości laboratoriów badawczych do celów akredytacji i okresowej kontroli, w szczególności przy braku próbek wzorcowych i przy niewielkiej liczbie elementów próbki oraz występowaniu danej odstającej. Dotyczy to w szczególności laboratoriów, które muszą przeprowadzać badania niszczące lub o wysokich kosztach pomiarów. Porównano na przykładach liczbowych oceny dokładności otrzymane proponowaną iteracyjną metodą odporną i według procedur standardowych.Advantages of robust iterative statistical method for estimating the accuracy of performance of testing laboratories during their accreditation in the absence of reference materials and with small sample sizes and outliers are presented in the paper. These situation is observed in the laboratory performing the test with the destruction of the samples or in the case of very expensive testing. A comparison with the estimates obtained by the standard procedure for evaluating performance accuracy is also provided

Glycated collagen induces a11 integrin expression through TGF-ß2 and Smad3

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the a11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates a11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased a11 integrin and a-smooth muscle actin expression. Compared with native collagen, binding of purified a11b1 integrin to glycated collagen was reduced by fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-b2 but not TGF-b1 or TGF-b3. The increased expression of TGF-b2 was inhibited by triple helical collagen peptides that mimic the a11b1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with a11 integrin luciferase promoter constructs, glycated collagen activated the a11 integrin promoter. Analysis of a11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between 809 and 1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-b2 signaling pathway that stimulates a11 integrin expression through Smad2/3 binding elements in the a11 integrin promoter, which is important for myofibroblast formation and fibrosis.This work was sponsored by the Canadian Institutes of Health Research

SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome

A novel autosomal recessive cerebro - renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of t he six affected individuals had also early - onset nephropathy with features of tubulo - interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified a ~ 18Mbs disease - associated locus on chro mosome 4 ( maximal logarithm of odds score 4.4 at D4S2971 ; θ =0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus , segregating as expected within the kindred and not found in a homozygous s tate in 300 Bedouin controls . We showed that SLC30A9 (Solute Carrier Family 30 , Member 9 ; ZnT - 9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH - SY5Y cells overexpressing SLC30A9 fused t o enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum , not co - localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signaling. However, using d ual - l uciferase r eporter a ssay in SH - SY5Y cells we showed that Wnt signaling was not affected by the mutation. Based on protein modeling, the iden tified mutation is expected to affect SLC30A9’s highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn 2+ measurements in HEK293 cells overexpressing wild - type and mutant SLC30A9 showed lower zinc concen tration within mutant rather than wild - type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through defective function of this novel activi ty of SLC30A9 rather than by a defect in its previously described role in transcriptional activation of Wnt signaling