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Tsou C.-C.,University of Michigan | Avtonomov D.,University of Michigan | Larsen B.,Lunenfeld Tanenbaum Research Institute | Tucholska M.,Lunenfeld Tanenbaum Research Institute | And 4 more authors.
Nature Methods | Year: 2015

As a result of recent improvements in mass spectrometry (MS), there is increased interest in data-independent acquisition (DIA) strategies in which all peptides are systematically fragmented using wide mass-isolation windows ('multiplex fragmentation'). DIA-Umpire (http://diaumpire.sourceforge.net/), a comprehensive computational workflow and open-source software for DIA data, detects precursor and fragment chromatographic features and assembles them into pseudo-tandem MS spectra. These spectra can be identified with conventional database-searching and protein-inference tools, allowing sensitive, untargeted analysis of DIA data without the need for a spectral library. Quantification is done with both precursor- and fragment-ion intensities. Furthermore, DIA-Umpire enables targeted extraction of quantitative information based on peptides initially identified in only a subset of the samples, resulting in more consistent quantification across multiple samples. We demonstrated the performance of the method with control samples of varying complexity and publicly available glycoproteomics and affinity purification-MS data. © 2015 Nature America, Inc.

PubMed | Barbara Ann Karmanos Cancer Institute, Palacky University, Washington State University, Aichi Cancer Center Research Institute and 34 more.
Type: | Journal: International journal of cancer | Year: 2017

It is not clear whether alcohol consumption is associated with lung cancer risk. The relationship is likely confounded by smoking, complicating the interpretation of previous studies. We examined the association of alcohol consumption and lung cancer risk in a large pooled international sample, minimizing potential confounding of tobacco consumption by restricting analyses to never smokers. Our study included 22 case-control and cohort studies with a total of 2548 never-smoking lung cancer patients and 9362 never-smoking controls from North America, Europe and Asia within the International Lung Cancer Consortium (ILCCO) and SYNERGY Consortium. Alcohol consumption was categorized into amounts consumed (grams per day) and also modelled as a continuous variable using restricted cubic splines for potential non-linearity. Analyses by histologic sub-type were included. Associations by type of alcohol consumed (wine, beer and liquor) were also investigated. Alcohol consumption was inversely associated with lung cancer risk with evidence most strongly supporting lower risk for light and moderate drinkers relative to non-drinkers (>0-4.9g per day: OR=0.80, 95% CI=0.70-0.90; 5-9.9g per day: OR=0.82, 95% CI=0.69-0.99; 10-19.9g per day: OR=0.79, 95% CI=0.65-0.96). Inverse associations were found for consumption of wine and liquor, but not beer. The results indicate that alcohol consumption is inversely associated with lung cancer risk, particularly among subjects with low to moderate consumption levels, and among wine and liquor drinkers, but not beer drinkers. Although our results should have no relevant bias from the confounding effect of smoking we cannot preclude that confounding by other factors contributed to the observed associations. Confounding in relation to the non-drinker reference category may be of particular importance. This article is protected by copyright. All rights reserved.

No statistical methods were used to predetermine sample size. YapΔ/Δ and TazΔ/Δ mice were generated by crossing Yap or Taz floxed mice30 with the villin-cre line (Jackson Laboratory), the villin-creERT2 line (S. Robine, Institut Curie-CNRS) or the Lgr5-creERT line (Jackson Laboratory). The Rosa26-lox-STOP-lox-rtta-IRES-EGFP and Rosa26 lacZ mouse lines were obtained from Jackson Laboratory. The YapTg transgenic line described in this study was generated by introducing a HA-tagged wild-type Yap cDNA downstream of 7 Tet-repressor elements in the pTRE2 vector (J. Whitsett, Cincinnati Children’s Hospital Medical Center). The transgenic Yap construct was linearized and microinjected in ICR embryos. As shown in Extended Data Fig. 3a, activation of Cre deletes a neo cassette and allows for expression of the rtTA gene. In the presence of doxycycline the rtTA activates transcription of HA-Yap. Apc floxed mice were obtained from O. Sansom (Beatson Institute). Lats1 and Lats2 floxed alleles were obtained from R. Johnson (MD Anderson Cancer Center) and crossed with villin-creERT2 mice to obtain Lats1Δ/Δ;Lats2Δ/Δ mice. To measure polyp formation, YapΔ/Δ mice were backcrossed to a Bl/6 background for 4 generations before crossing to ApcMin/+ mice. Polyps from Yap+/+ (Yap+/+;villin-cre;ApcMin/+), Yap+/Δ (Yapfl/+;villin-cre;ApcMin/+) and YapΔ/Δ (Yapfl/fl;villin-cre;ApcMin/+) mice were counted 16 weeks after birth or when animals appeared moribund. Survival of ApcMin mice was measured by the number of days before mice were euthanized due to poor health. In vivo assays comparing control and Yap mutant animals were performed between age- and sex-matched pairs. No method of randomization was followed and no animals were excluded in this study. The investigators were not blinded to allocation during experiments and outcome assessment. Inducible Cre-mediated deletion of genes was performed by intraperitoneal injections of >5-week-old mice with 200 μl tamoxifen in corn oil at 10 mg ml−1. To create mosaic expression of Yap, Yapfl/fl;villin-creERT2 mice were induced with a single injection of 200 μl of tamoxifen at a suboptimal dose typically between 0.5 and 2.0 mg ml−1. For in vivo regeneration assays, mice were given a single dose of 10 or 12 Gy using a GammaCell 40 irradiator. Animals were maintained and handled under procedures approved by the Canadian Council on Animal Care. The immunohistochemistry stainings and standard colorimetric in situ hybridization were carried out according to methods described elsewhere31. Staining experiments were repeated on independent tissue sections prepared from separate mice as indicated by n values in figure legends. The following primary antibodies were used for immunostaining: rat anti-Ki67 (Dako, Cat. no. M7249, 1:1,000), rabbit anti-Yap/Taz (Cell Signaling, Cat. no. 8418, 1:100), rabbit anti-Yap (Cell Signaling, Cat. no. 14074, 1:300), mouse anti-Yap (Santa Cruz, Cat. no. sc-101199, 1:100), rabbit anti-Lef (Cell Signaling, Cat. no. 2230, 1:300), phosphor-Egfr (Tyr1092) (Abcam, Cat. no. ab40815, 1:300), anti-cleaved caspase-3 (Cell Signaling, Cat. no. 9664, 1:300) and anti-lysozyme (Dako, Cat. no. A0099, 1:1,000). Detection of primary antibodies was achieved using the Dako Envision plus system. Multi-colour fluorescence in situ hybridization with tyramide signal amplification (TSA) was done essentially as described elsewhere32, 33, 34. In brief, RNA probes from hybridized sections were detected using appropriate hapten-specific HRP-conjugated antibodies (anti-digoxigenin-HRP (Roche, Cat. no. 11207733910, 1:500), anti-dinitrophenyl-HRP (PerkinElmer, Cat. no. NEL747A001KT, 1:300), and anti-fluorescein-HRP (Life Technologies, A21253, 1:500)). After overnight incubation with antibodies at 4°C (or 2 h at room temperature for anti-fluorescein-HRP detection of cryptin1) sections were washed in PBS, and rinsed twice in 100 mM borate pH 8.5 plus 0.1% BSA. TSA reaction was performed by applying 300 μl per slide of the following mixture: 100 mM borate pH 8.5, 2% dextran sulfate, 0.1% Tween-20 and 0.003% H O , 450 μg ml−1 4-iodophenol: 1:250 Tyramide product (that is, DyLight633-tyramide, Dylight488-tyramide, Dylight 555-tyramide). The TSA reaction was allowed to proceed for 20 min and then terminated by washing slides in 100 mM glycine pH 2.0 for 15 min. Sections were washed further in PBS for the next round of detection. To synthesize tyramide products, the following succinimidyl esters were used for conjugation with tyramine: DyLight 633 NHS-Ester (Thermo Scientific Cat#46414), DyLight 550 NHS-Ester (Thermo Scientific Cat#62262), DyLight 488 NHS-Ester (Thermo Scientific Cat. no. 46402). The synthesis reaction was carried out as described previously32. The following in situ hybridization probes were obtained from the collection of MGC clones at the Lunenfeld Tanenbaum Research Institute: TweakR (BC025860), Ly6c1 (BC092082), Edn1 (BC029547), Areg (BC009138), Ereg (BC027838), Il1rn (BC042532), Il33 (BC003847), Msln (BC023753) and Cyr61 (BC066019). The Olfm4 and cryptdin1 probes were a gift from H. Clevers (Hubrecht Institute). Before fixing organoids, 10 μM Edu was added to the culture media for 1 h. Then organoids were fixed in 10% buffered formalin for 30 min, permeabilized in 0.5% Triton for 20 min and blocked in 2% BSA. Incorporated Edu was detected using the ClickIt EDU Imaging kit (Invitrogen) according to the manufacturer’s instructions. The primary antibodies used for immunostaining were mouse anti-Yap (Santa Cruz, Cat # sc-101199, 1:100), mouse anti-HA (Sigma-Aldrich, Cat. no. H9658, 1:1,000), and chicken anti-β-gal (Abcam, Cat. no. ab9361, 1:300). The secondary antibodies used in immunostaining were: CF555-donkey anti-mouse (Biotium, Cat. no. 20037, 1:400) and CF647 donkey anti-rabbit (Biotium, Cat. no. 20047, 1:400). Organoids were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich) before mounting onto slides for visualization. Images were acquired using a 20×/NA oil immersion objective lens (HCX PL APO, Leica), an EM-CCD camera (ImagEM, Hamamatsu) on an inverted microscope (DMIRE2, Leica) with a spinning disk confocal scanner (CSU10, Yokogawa) and Volocity. De novo crypts were scored as any protrusions, typically containing Paneth cells, budding from the initial sphere formed after seeding isolated crypts. Crypts were counted from bright-field images using Image J. At least four independent cultures derived from four different mice per genotype were used for quantification. Survival of crypts in Fig. 1 was determined by Ki67 staining of cross-sections of proximal portions of the small intestine at 3 days post-irradiation (10 Gy or 12 Gy). Values in Fig. 1b represent average number of fully labelled Ki67+ crypts per intestinal circumference based on counts from at least two sections per mouse and assays were repeated in 6 independent mice per genotype for both 10 Gy and 12 Gy treatments. The percentage of surviving Yap-positive versus negative Lgr5+ ISCs in Fig. 1d was performed by counting 587 β-gal+ crypts from a total of 7 untreated YapΔLgr5-cre mice and 394 β-gal+ crypts from a total of 9 irradiated YapΔLgr5-cre mice. In Yap;ApcΔLgr5-cre mice tumour initiating cells were visualized by staining for the Wnt target gene, Lef. As shown in Extended Data Fig. 10b, Lef is undetected in wild-type crypts and highly upregulated in Apc-null cells and thus serves as a robust marker of Apc deletion31. The percentage of Paneth cells in Lef+ foci (Fig. 4a) was assessed by preparing consecutive sections stained for Lyz, Lef and Yap, respectively. Lysozyme-positive Paneth cells from a total of 207 Yap wild-type and 201 Yap mutant Lef+ foci were counted from 5 Yap;ApcΔLgr5-cre mice (10–16 days after tamoxifen injection) using Image J and the percentage of total cells within the boundaries of a given Lef+ lesion was calculated. Relative activation of Egfr was quantified in consecutive sections from 5 Yap;ApcΔLgr5-cre mice stained for Lef, Yap and phospho-Egfr. For assessing Phospho-Egfr, staining intensity in Lef+ foci was assessed in a blinded fashion. For this, consecutive sections stained for Yap were masked from the observer scoring phospho-Egfr staining intensity. Lef+ foci were scored as ‘+’ if phospho-Egfr expression was elevated compared to wild-type adjacent crypts at comparable levels within the crypt–villus axis (see Extended Data Fig. 10f, panels xi and xii). Lef+ foci were scored as ‘++’ if staining intensity was very strong even relative to the stem cell compartment in normal crypts and/or displayed prominent apical staining (see Yap-positive foci in Fig. 4c, panel iv, and Extended Data Fig. 10f, panels v and vi). Lef+ foci were scored as ‘–’ if staining intensity was undetected or unchanged relative to adjacent wild-type crypts (see Yap mutant foci in Fig. 4c and Extended Data Fig. 10f). In Extended Data Fig. 1, caspase 3 and BrdU positive crypt cells were counted from at least six sections per mouse in 4 independent mice per genotype and expressed as a percentage of total crypt cells. All data are presented as average values with s.e.m. Mann–Whitney (two-tailed) U-test was used to determine statistical significance. Calculations were performed using GraphPad Prism 5 software. RNA was isolated from organoids cultured for 24 h after seeding in Matrigel. RNA samples were pooled from at least three organoid cultures derived from at least three independent mice per genotype (Yapfl/+;villin-cre, Yapfl/fl;villin-cre and YapTg). Quality of RNA was verified by running samples on a Bioanalyzer. High-throughput sequencing was performed using the Illumina HiSeq 2000 at the Lunenfeld Tanenbaum Research Institute (LTRI) sequencing facility. Raw sequencing reads in Fastq formats were mapped onto mouse genome (mm9) using Tophat 1.4.1 and the RPKMs (reads per kilobase of exon model per million mapped reads) were calculated using a customized script. RNA-seq data are presented in Supplementary Table 1. Combined fold change presented in Extended Data Fig. 3d and Supplementary Table 1 was calculated using the following formula: combination fold change = log [(YapΔ/Δ/Yap+/Δ)/(Dox+/Dox−)]. R, Cluster 3.0 and Java TreeView were used for data visualization. Gut organoids were cultured according to a previously described protocol established by Sato and Clevers7. Briefly, crypts were harvested by incubating opened small intestines in PBS containing 2 mM EDTA. The epithelium was released by vigorous shaking and crypts separated using a 70 μm cell strainer. Crypts were seeded in growth factor reduced Matrigel (BD Biosciences) and grown in Advanced DMEM/F12 (Invitrogen) supplemented with 2 mM GlutaMax (Invitrogen), 100 U ml−1 Penicillin/100 μg ml−1 Streptomycin (Invitrogen), N2 Supplement (Invitrogen), B-27 Supplement (Invitrogen Cat), mouse recombinant Egf (R&D Systems), 100 ng ml−1 mouse recombinant Noggin (Peprotech), 150 ng ml−1 human Rsp1 (R&D Systems). Apc-deficient organoids were harvested from Yapfl/+;Apcfl/fl;villin-creERT, Yapfl/fl;Apcfl/fl;villin-creERT or YapTg;Apcfl/fl;villin-creERT mice injected with tamoxifen and seeded 48 h later in basal growth medium without Egf, Rsp1 or Noggin. To induce Yap expression in YapTg organoids, 1.5 μg ml−1 doxycycline was added to the culture medium on day 0. Egf (R&D Systems, Cat. no. AF2028), Areg (R&D Systems, Cat. no. AF989) and Ereg (R&D Systems, Cat. no. 1068-EP-050) were added to the culture medium at a final concentration of 0.5 μg ml−1. The following inhibitors were used: PD153053 (0.5 μM, Tocris Bioscience), U0126 (10 μM, Merck Millipore). To examine pErk1/2 levels, organoids were harvested at day 2 in cold PBS containing 5 mM EDTA, 1 mM NaVO , 1.5 mM NaF and protease inhibitors. Organoids were incubated at 4°C for 30 min to dissolve Matrigel and then lysed in TNTE buffer (50 mM Tris/HCl pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA) containing standard protease and phosphatase inhibitors. Protein concentrations were measured and samples were subjected to SDS–PAGE. Total RNA was extracted by removing culture medium and directly lysing organoids in wells using RTL buffer of the Rneasy Mini Kit (Qiagen). RNA was purified using columns and genomic DNA was removed by treatment with RNase-Free DNase (Qiagen).

News Article | November 22, 2016
Site: www.marketwired.com

Researchers at Sinai Health System's Lunenfeld-Tanenbaum Research Institute focus on genetic and environmental factors TORONTO, ON--(Marketwired - November 22, 2016) - Scotiabank has announced a $1 million gift to one of the top biomedical research institutes in North America to support discovery of the determinants of health for infants and children. The announcement was made on Monday, November 21, at Sinai Health Foundation's marquee event Dinner with Scientists. The gift will support the work of Dr. Stephen Lye, a Senior Investigator at the Lunenfeld-Tanenbaum Research Institute, part of Sinai Health System. Dr. Lye's research focus is to better understand both the genetic and environmental factors that shape the health of infants and children, from the time of conception. He has published studies on such topics as the impact of breastfeeding, pre-term birth and placental health. He is also co-lead of the Ontario Family Health Study, which is collecting data from over a thousand pregnant women and, through TARGet Kids, following the health of their children. Dr. Lye holds the Mount Sinai Hospital Auxiliary Chair in Women's and Infants' Health Research. Sinai Health System has the largest neonatal research program in Canada -- based on activity, volume of research and funding. "The goal of this research is to translate our findings into public health and education policy, as well as paediatric clinical practice. By supporting this research, Scotiabank is contributing to better outcomes for our children's future," said Dr. Lye. The gift will be matched by the Tanenbaum Research Matching Endowment Fund, which was created by Larry and Judy Tanenbaum and their family. The fund provides 1:1 match funding for gifts of $1 million from other donors, providing donors with the opportunity to double their impact in supporting world-leading science. "We are honoured to partner with Dr. Lye in supporting his important work to improve the lives of infants and children, as well as their families," says Brian Porter, President and CEO of Scotiabank. "Health is fundamental to our young people reaching their full potential -- there are few other investments as important to the long-term security, stability and strength of our communities and our country." "We have had a long partnership with Scotiabank for over 60 years and they have been strong advocates in supporting discovery science at the LTRI," says Joseph Mapa, CEO of Sinai Health Foundation. "A gift of this nature provides critical funding that helps us prevent, diagnose and treat the conditions and diseases that affect Canadians. We thank them for their generous support." The Lunenfeld-Tanenbaum Research Institute, part of Sinai Health System, is a leading biomedical research centre, ranking amongst the top biomedical research institutes in the world. Established in 1985, the institute is profoundly advancing understanding of human biology in health and disease. Many of the breakthroughs that began as fundamental research have resulted in new and better ways to prevent, diagnose and treat prevalent conditions. The institute is affiliated with the University of Toronto and is focused on women's and infants' health, cancer biology, stem cell biology, neurobiology, diabetes, arthritis, health systems research, population health services and solutions, and systems biology. www.lunenfeld.ca. Through our global community investment strategy, Scotiabank and its employees support causes at a grassroots level. Recognized as a leader for our charitable donations and philanthropic activities, in 2015, Scotiabank contributed $67 million to help our communities around the world. Scotiabank is Canada's international bank and a leading financial services provider in North America, Latin America, the Caribbean and Central America, and Asia-Pacific. We are dedicated to helping our 23 million customers become better off through a broad range of advice, products and services, including personal and commercial banking, wealth management and private banking, corporate and investment banking, and capital markets. With a team of more than 88,000 employees and assets of $907 billion (as at July 31, 2016), Scotiabank trades on the Toronto (TSX: BNS) and New York Exchanges ( : BNS). Scotiabank distributes the Bank's media releases using Marketwired. For more information, please visit www.scotiabank.com and follow us on Twitter @ScotiabankViews.

INGELHEIM, Germany & INDIANAPOLIS, US--(BUSINESS WIRE)--New data presented at the American Heart Association Scientific Sessions 2016 showed that Jardiance® (empagliflozin) consistently reduced the risk for cardiovascular death, regardless of the type of cardiovascular disease at baseline, compared with placebo when added to standard of care in adults with type 2 diabetes and established cardiovascular disease. The findings are part of the landmark EMPA-REG OUTCOME® trial, which is the first trial of a diabetes medication to show a reduction in cardiovascular death in adults with type 2 diabetes and established cardiovascular disease. This study is supported by Boehringer Ingelheim and Eli Lilly and Company (NYSE: LLY). “Cardiovascular disease is two to four times more common in people with type 2 diabetes and is an umbrella term consisting of several different vascular outcomes, including heart attack, heart failure, peripheral vascular disease and stroke,” said lead investigator of the trial Dr Bernard Zinman, Director, Diabetes Centre, Mount Sinai Hospital, Toronto; Senior Scientist, Lunenfeld Tanenbaum Research Institute, and Professor of Medicine, University of Toronto, Canada. “Since approximately 50 percent of deaths in people with type 2 diabetes worldwide are attributed to cardiovascular causes, we need diabetes therapies that help reduce this complication in individuals who have an underlying cardiovascular issue.” For this post hoc analysis, trial participants were grouped based on type of cardiovascular disease at baseline, which included history of heart attack, stroke, heart failure, atrial fibrillation and existing peripheral artery disease. Lower rates of cardiovascular death were seen in the Jardiance® group independent of cardiovascular disease type. Observed adverse events were consistent with the known safety profile of Jardiance®. “Jardiance is the only oral type 2 diabetes medicine shown in a clinical trial to reduce the risk of cardiovascular death,” said Professor Hans-Juergen Woerle, Global Vice President Medicine, Metabolism, Boehringer Ingelheim. “These results provide further evidence and reinforce the strength of the EMPA-REG OUTCOME data demonstrating a reduction in risk of cardiovascular death in adults with type 2 diabetes and established cardiovascular disease.” EMPA-REG OUTCOME® was a long-term, multicentre, randomised, double-blind, placebo-controlled trial of more than 7,000 patients, from 42 countries, with type 2 diabetes and established cardiovascular disease. The study assessed the effect of Jardiance® (10 mg or 25 mg once daily) added to standard of care compared with placebo added to standard of care. Standard of care was comprised of glucose-lowering agents and cardiovascular drugs (including for blood pressure and cholesterol). The primary endpoint was defined as time to first occurrence of cardiovascular death, non-fatal heart attack or non-fatal stroke. Over a median of 3.1 years, Jardiance® significantly reduced the risk of cardiovascular death, non-fatal heart attack or non-fatal stroke by 14 percent versus placebo. Risk of cardiovascular death was reduced by 38 percent, with no significant difference in the risk of non-fatal heart attack or non-fatal stroke. The safety profile of Jardiance® in the EMPA-REG OUTCOME® trial was consistent with that of previous trials. The overall incidence of adverse events was similar to placebo. More than 415 million people worldwide have diabetes, of which 193 million are estimated to be undiagnosed. By 2040, the number of people with diabetes is expected to rise to 642 million people worldwide. Type 2 diabetes is the most common form of diabetes, responsible for up to 91 percent of diabetes cases in high-income countries. Diabetes is a chronic condition that occurs when the body either does not properly produce, or use, the hormone insulin. Due to the complications associated with diabetes, such as high blood sugar, high blood pressure and obesity, cardiovascular disease is a major complication and the leading cause of death associated with diabetes. People with diabetes are two to four times more likely to develop cardiovascular disease than people without diabetes. In 2015, diabetes caused 5 million deaths worldwide, with cardiovascular disease as the leading cause. Approximately 50 percent of deaths in people with type 2 diabetes worldwide are caused by cardiovascular disease. Jardiance® (empagliflozin) is an oral, once daily, highly selective sodium glucose co-transporter 2 (SGLT2) inhibitor approved for use in Europe, the United States and other markets around the world for the treatment of adults with type 2 diabetes. Jardiance® works by blocking the reabsorption of glucose (blood sugar) by the kidney, leading to urinary glucose excretion, and lowering blood glucose levels in people with type 2 diabetes. SGLT2 inhibition targets glucose directly and works independently of β-cell function and the insulin pathway. Jardiance® is not for people with type 1 diabetes or for people with diabetic ketoacidosis (increased ketones in the blood or urine). This press release is issued from Boehringer Ingelheim Corporate Headquarters in Ingelheim, Germany and is intended to provide information about our global business. Please be aware that information relating to the approval status and labels of approved products may vary from country to country, and a country-specific press release on this topic may have been issued in the countries where Boehringer Ingelheim and Eli Lilly and Company do business. This press release contains forward-looking statements (as that term is defined in the Private Securities Litigation Reform Act of 1995) about JARDIANCE as a treatment of adults with type 2 diabetes and established cardiovascular disease, and reflects Lilly’s current belief. However, as with any pharmaceutical product, there are substantial risks and uncertainties in the process of development and commercialization. Among other things, there can be no guarantee that future study results will be consistent with the results to date or that JARDIANCE will receive additional regulatory approvals. For further discussion of these and other risks and uncertainties, see Lilly's most recent Form 10-K and Form 10-Q filings with the United States Securities and Exchange Commission. Except as required by law, Lilly undertakes no duty to update forward-looking statements to reflect events after the date of this release. Please click on the link below for ‘Notes to Editors’ and ‘References’:

Janouskova E.,Masaryk University | Necasova I.,Masaryk University | Pavlouskova J.,Masaryk University | Zimmermann M.,Masaryk University | And 5 more authors.
Nucleic Acids Research | Year: 2015

More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex. © The Author(s) 2015.

Gunaratne A.,University of Western Ontario | Gunaratne A.,Lunenfeld Tanenbaum Research Institute | Chan E.,University of Western Ontario | El-Chabib T.H.,University of Western Ontario | And 3 more authors.
Journal of Cell Science | Year: 2015

Transforming growth factor β (TGFβ) signaling controls many cellular responses including proliferation, epithelial to mesenchymal transition and apoptosis, through the activation of canonical (Smad) as well as non-canonical (e.g. Par6) pathways. Previous studies from our lab have demonstrated that aPKC inhibition regulates TGFβ receptor trafficking and signaling. Here, we report that downstream TGFβ-dependent transcriptional responses in aPKC-silenced NSCLC cells were reduced compared with those of control cells, despite a temporal extension of Smad2 phosphorylation. We assessed SARA-Smad2-Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA-Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2-Smad4 complex formation, which correlated with reduced Smad2 nuclear translocation. Interestingly, we also detected an increase in p38 MAPK phosphorylation and apoptosis in aPKC-silenced cells, which were found to be TRAF6-dependent. Taken together, our results suggest that aPKC isoforms regulate Smad and non-Smad TGFβ pathways and that aPKC inhibition sensitizes NSCLC cells to undergo TGFβdependent apoptosis. © 2015. Published by The Company of Biologists Ltd.

Gingras A.-C.,Lunenfeld Tanenbaum Research Institute | Gingras A.-C.,University of Toronto | Wong C.J.,Lunenfeld Tanenbaum Research Institute | Wong C.J.,University of Toronto
Current Opinion in Structural Biology | Year: 2016

Cells have evolved intricate ways to propagate signals through signaling networks rich in crosstalks between signaling pathways and feedforward and feedback loops. The enzymatic products of phosphorylation-dependent signaling, namely phosphopeptides, can be identified and quantified systematically by mass spectrometry. Recent advances in the speed and sensitivity of mass spectrometers, combined with improvements in sample preparation and data analysis, are enabling the acquisition of increasingly large phosphopeptide datasets. Here, we discuss several considerations in experimental design that aid in unraveling direct and indirect phosphorylation events, as well as to identify crosstalks and feedback loops. © 2016 Elsevier Ltd

PubMed | Li Ka Shing Knowledge Institute, Lunenfeld Tanenbaum Research Institute, Brock University, Quality and Chief Nurse Executive Sinai Health System and 4 more.
Type: | Journal: Worldviews on evidence-based nursing | Year: 2017

Nursing plays a central role in facilitating care transitions for complex older adults, yet there is no consensus of the components of nurse-led care transitions interventions to facilitate high quality care transitions among complex older adults. A structured expert panel was established with the purpose of identifying effective nurse-led care transition interventions.A modified Delphi consensus technique based on the RAND method was employed. Panelists (n = 23) were asked to individually rate a series of statements derived from a realist synthesis of the literature for relevance, feasibility and likely impact. Statements receiving an aggregate score of 75% (7/9) were reviewed and revised at a face-to-face consensus meeting. A second round of rating following the same process as round one was used, followed by a final ranking of the statements.The five highest ranked intervention components and contextual factors were: (a) educating and coaching patients, their family members and caregivers about self-management skills; (b) ensuring patients, their family members and caregivers are aware of follow-up medical appointments and postdischarge care plan; (c) using standardized documentation tools and comprehensive communication strategies during care transitions; (d) optimizing nurses roles and scopes of practice across the care transitions spectrum; and (e) having strong leadership, strategic alignment and accountability structures in organizations to enable quality care transitions for the complex older person population.Key insights on optimizing the nurses roles and scope of practice during care transitions included having nurses provide warm hand-offs and serve as the go-to person. The panel also identified current challenges to optimizing the nurses roles and scope of practice across care transition points. Future research is required to determine effective nurse-led intervention components and in which context do they work or do not.

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