Reporter constructs with low background activity utilizing the cat gene

Reporter plasmids utilizing the cat gene for the analysis of promoter and enhancer sequences in vertebrate cells, were constructed. These plasmids minimize the background of transcription derived from cryptic promoters or cryptic regulatory elements within the vecto

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Analysis of regulatory W mutations and the function of the Kit receptor tyrosine kinase in the intestine

grantor: University of TorontoThe murine 'W' locus encodes the Kit receptor tyrosine kinase (RTK). Many structural mutations at the 'W' locus, affecting the Kit coding sequence, lead to pleiotropic defects in hematopoiesis, gametogenesis and melanogenesis. Regulatory 'W' mutations, on the other hand, do not affect the Kit coding sequence and cause cell type specific defects. Moreover, the Kit RTK is expressed in several cell types and tissues with no obvious defects in 'W' mutant mice, raising the question of the function of the Kit molecule in these tissues. In this thesis, I have analyzed the molecular basis and developmental defects associated with two spontaneous regulatory mutations in the mouse ' W' locus. I describe the effects of the 'W57' and Wbandedbd mutations on 'Kit' expression during embryogenesis and in the adult animal and show specific defects in melanocyte development in these mutant animals. In addition, I describe the molecular basis of these regulatory 'W' mutations. The Kit RTK is expressed in the adult intestine, but no intestinal defects had been described in 'W' mutant animals. I identify the Kit-expressing cells in the small intestine as the interstitial cells of Cajal (ICC) and demonstrate the importance for this cell type in intestinal pacemaking. Moreover, I analyze the developmental origin and lineage relationships of these Kit-expressing cells in the small intestine and investigate the role of Kit in their development. ICC and smooth muscle cells of the intestine are derived from a common precursor. Moreover, a functional Kit receptor is not required as an instructive signal during lineage determination, but is necessary for ICC proliferation after birth. These results provide novel information on the multiple roles of the Kit RTK in mouse development, as well as new insights into the regulation of expression of this gene.Ph.D

Chondroitin Sulfate-E Is a Negative Regulator of a Pro-Tumorigenic Wnt/Beta-Catenin-Collagen 1 Axis in Breast Cancer Cells

<div><p>Expression of the glycosaminoglycan chondroitin sulfate-E (CS-E) is misregulated in many human cancers, including breast cancer. Cell-surface associated CS-E has been shown to have pro-tumorigenic functions, and pharmacological treatment with exogenous CS-E has been proposed to interfere with tumor progression mediated by endogenous CS-E. However, the effects of exogenous CS-E on breast cancer cell behavior, and the molecular mechanisms deployed by CS-E are not well understood. We show here that treatment with CS-E, but not other chondroitin forms, could interfere with the invasive protrusion formation and migration of breast cancer cells in three-dimensional organotypic cultures. Microarray analysis identified transcriptional programs controlled by CS-E in these cells. Importantly, negative regulation of the pro-metastatic extracellular matrix gene <i>Col1a1</i> was required for the anti-migratory effects of exogenous CS-E. Knock-down of <i>Col1a1</i> gene expression mimics the effects of CS-E treatment, while exposing cells to a preformed collagen I matrix interfered with the anti-migratory effects of CS-E. In addition, CS-E specifically interfered with Wnt/beta-catenin signaling, a known pro-tumorigenic pathway. Lastly, we demonstrate that <i>Col1a1</i> is a positively regulated target gene of the Wnt/beta-catenin pathway in breast cancer cells. Together, our data identify treatment with exogenous CS-E as negative regulatory mechanism of breast cancer cell motility through interference with a pro-tumorigenic Wnt/beta-catenin - Collagen I axis.</p></div

CS-E reduces 3D invasive protrusion formation and migratory behavior of EMT6 cells.

<p>(<b>A</b>) EMT6 cells grown in 3D on-top Matrigel assays were treated with 100 microgram/ml C4S, C6S, CS-D, CS-E, or no treatment control conditions for 6 days, and assessed by live phase contrast microscopy (scale bar, 100 micrometer). At day 6, the percent of 3D structures containing 5 or more invasive protrusions were quantified for each condition. Arrows represent invasive protrusions on 3D structures. (<b>B</b>) Inhibition of invasive protrusion formation by CS-E treatment is concentration dependent. EMT6 cells were treated in control conditions or with increasing amounts of CS-E for 6 days in 3D on-top Matrigel conditions. (<b>C</b>) EMT6 cell 3D cultures were grown in control conditions or with 100 microgram/ml CS-E for 6 days with EdU addition 1 hour prior to fixation for detection of proliferating cells by confocal microscopy. (<b>D, E</b>) Invasion/migration transwell assays. EMT6 cells were plated on migration control inserts (<b>D</b>), or on BD BioCoat invasion chambers coated with growth factor reduced Matrigel (<b>E</b>) and treated with or without 100 microgram/ml of CS-E for 24 hours. Total cell counts show that CS-E treatment reduced EMT6 cell migration by 2.3 fold, but did not change cell invasion. (n = 6). (*p<0.01).</p

Negative regulation of Col1a1 expression by CS-E is required for inhibition of cell migration in EMT6 and 4T1 breast cancer cells.

<p>(<b>A</b>) Knockdown of Col1a1: <i>Col1a1</i> mRNA levels were compared by qRT-PCR after 24 hrs of either Col1a1 siRNA (siCol1a1) or non-targeting siRNA control (siCon) knockdown. Col1a1 siRNA knockdown reduced <i>Col1a1</i> expression to approximately 25% in both EMT6 and 4T1 cells. (<b>B</b>) Western blot analysis of the effect of siCol1a1 or siCon on secreted Col1 protein levels. Transfection of siCol1a1 reduces levels of secreted type 1 collagen in both EMT6 and 4T1 cells. (<b>C</b>) siRNA knockdown of Col1a1 significantly reduces breast cancer cell motility in 24 hour transwell experiments. (<b>D</b>) Establishing an exogenous collagen I matrix interfered with the inhibitory effects of CS-E on cell migration. EMT6 or 4T1 cells were plated onto migration inserts that were uncoated or coated with purified bovine type 1 collagen and were treated with or without 100 microgram/ml CS-E for 8 hours in a transwell migration assay. CS-E could interfere with cell migration in the absence, but not the presence of an exogenously supplied collagen I matrix. Graph shows average number of migrating cells per experiment (n = 3). (*p<0.05; ns = not significant).</p

<i>Col1a1</i> is a Wnt/beta-catenin target gene in breast cancer cells.

<p>(<b>A</b>) RTqPCR to quantify <i>Col1a1</i> mRNA expression in response to Wnt3a stimulation, in the presence or absence of CS-E. Treatment with Wnt3a-CM led to a 2-fold (EMT6) and 4-fold (4T1) increase in <i>Col1a1</i> mRNA expression. Concomitant treatment with CS-E significantly interfered with this stimulatory effect of Wnt3a and reduced <i>Col1a1</i> expression levels (*p<0.05). (<b>B</b>) Wnt/beta-catenin pathway inhibition by IWR-1. IWR-1 at both 5 microMolar and 15 microMolar completely inhibited Wnt3a-stimulated TOPFLASH activity to the level of control treated cells in EMT6 cells. In 4T1 cells, an IWR-1 concentration of 40 microM led to an almost complete loss of TOPFLASH activity. (<b>C</b>) RTqPCR analysis: inhibition of Wnt/beta-catenin signaling by IWR-1 (EMT6 cells: 5 microMolar; 4T1: 40 microMolar) led to a loss of Wnt3a-mediated induction of <i>Col1a1</i> mRNA expression in EMT6 and 4T1 cells.</p

Effect of curing conditions on the water vapor sorption behavior of melamine formaldehyde resin and resin-modified wood

Impregnation modification of wood with melamine formaldehyde resin reduces the adverse effects caused by moisture uptake, but the underlying modes of action are not fully understood. The present study showed that it is crucial to understand the sorption behavior of the pure resin when interpreting the behavior of resin-modified wood. Furthermore, the applied heat-curing conditions had a significant effect on the moisture uptake of resin-modified wood. At the same resin loads, dry curing conditions were more effective in causing a cell wall bulking effect than wet curing conditions. This reduced the water-accessible cell wall pore volume in dry cured wood and counterbalanced the moisture uptake by the resin. Deuterium exchange measurements suggested that the occupancy of cell wall pores reduced the number of simultaneously active sorption sites. However, there was no evidence that a swelling restraint or reduced mechanical relaxation affected the water sorption of resin-modified wood significantly.Peer reviewe

Microarray results of the effects of CS-E on gene expression profiles of EMT6 3D structures.

<p>Shown are fold-changes in mRNA expression (≥1.8-fold induction/repression). Two pro-tumorigenic Collagen genes (<i>Col1a1</i>, <i>Col6a2</i>) were repressed by CS-E treatment.</p