Localization and dynamics of homeotic oncogenic protein HOXC13 in pre-initiation complex of human DNA replication origins

In metazoan cells the DNA replication origins are not well defined. Differently from what observed for bacteria cells and for budding yeast, in metazoan the origins does not show a conserved sequence and they appear to be specified by many factors. In order to better understand the mechanisms involved in the origin specification, many studies have been done to identify the proteins involved in the recognition and activation of the origins. From these kind of analysis is emerging that, beside the wellknown proteins of the pre replicative complex, also other factors might be involved. Between these, the HOX proteins seem to be able to play a role in the origin activity. One of the first studies of this involvement was done by our group and leads to the identification of three homeotic proteins able to specifically bind in vitro the human lamin B2 origin. Thus, in the study conducted during this PhD program, was investigated the involvement of one of these homeotic proteins, namely HOXC13, with human DNA replication origins and with replicative complexes. We found an interaction of HOXC13 with two crucial factors of the pre Replication Complex (pre-RC), ORC1 and Cdc6 and that HOXC13 binds a good fraction of the origins, in particular the early replicating ones, like the lamin B2 origin and other known human origins. The HOXC13 protein is bound to origin chromatin, at least for the lamin B2 origin, at a precise site within the pre-RC at specific moments of the cell cycle. Interaction with the origin occurs within the area protected by the pre-RC in G1, very close to the start sites of leading strand synthesis and to the binding sites of ORC1, ORC2, Cdc6, topoisomerase (topo) I and topo II. The protein is absent from the origin in M and appears on it at the beginning of G1, reach a peak at G1/S and as synthesis starts, the interaction of HOXC13 with the origin fades, in parallel with the transition from this large pre-RC to a smaller and differently organized post-RC. Recently also other HOX proteins have been identify as proteins involved in regulation processes of DNA replication, suggesting that the interaction of HOXC13 with the origins might occur in a multi-homeotic proteins complex. Depletion of one of these proteins however is compatible with the continuation of the cell cycle and, according with what observed for the other homeotic proteins, we found that also the depletion of HOXC13 does not alter cell cycle progression or S phase entry. This is probably due to the redundancy of homeotic proteins and indicates a relatively generic function for the HOX proteins. Among the identified elements influencing the choice and the activity of a sequence as DNA replication origin, much relevance is assumed by the chromatin structure and topology of DNA. Therefore, we analysed the effects of chromatin structure disruption using Tricostatin A, a histone deacetylase inhibitor. The alteration of chromatin caused by this treatment not only sharply reduces origin function, but also disturbs the binding of replication complex members like HOXC13 and the well known Cdc6 to the DNA replication origins, while does not affect the binding of other unrelated proteins like USF1. On the basis of this finding, we infer that an appropriate chromatin organization and DNA topology strongly influence the binding between factors of the pre Replication Complex and DNA replication origins. This influence could be a key element in origin specification. The described interactions are not restricted to a single origin nor to a single homeotic protein, leading us to conclude that HOX proteins, probably in the context of a multi-protein homeotic effectors, contribute to recruit and stabilize the replicative complexes onto early replicating origins, in presence of specific chromatin and topological configurations. The relevance of HOXC13 in DNA replication is also underlined by its involvement in oncogenesis, clearly demonstrated in acute myeloid leukaemia when HOXC13 is fused with NUP98 protein

The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations

First principles study of the optical emission of cadmium yellow: Role of cadmium vacancies

We study the role of structural defects in the CdS-based cadmium yellow paint to explain the origin of its deep trap states optical emission. To this end, we combine a first principles study of Cd- and S- vacancies in the wurtzite (1010) CdS surface with experimental photoluminescence spectroscopy of the commercial hexagonal CdS pigment. Computational results clearly state that the presence of cadmium vacancies in the pigment surface alters the electronic structure of cadmium sulfide by forming acceptor levels in the gap of the semiconductor. Such levels are consistent with the optical emission from trap state levels detected in the CdS pigment. This finding provides a first step towards the understanding of the photo-physical mechanisms behind the degradation of this modern pigment, widely used in impressionist and modernist paintings

Integration of Biomechanical and Biological Characterization in the Development of Porous Poly(Caprolactone)-Based Membranes for Abdominal Wall Hernia Treatment.

AIMS: Synthetic meshes are the long-standing choice for the clinical treatment of abdominal wall hernias: the associated long-term complications have stimulated the development of a new-generation of bio-resorbable prostheses. In this work, polycaprolactone (PCL) porous membranes prepared by solvent casting/porogen leaching of PCL/poly(ethylene glycol) (PEG) blends with different compositions (different PCL/PEG weight ratio and PEG molecular weight) were investigated to be applied in the field. An optimal porous membrane structure was selected based on the evaluation of physicochemical, biomechanical and in-vitro biological properties, compared to a reference commercially available hernia mesh (CMC). FINDINGS: Selected PCL7-2i membranes (derived from PCL/PEG 70/30, PCL: Mw 70,000-90,000 Da; PEG: 35,000 Da) showed suitable pore size for the application, intermediate surface hydrophilicity and biomimetic mechanical properties. In-vitro cell tests performed on PCL7-2i membranes showed their cytocompatibility, high cell growth during 21 days, a reduced production of pro-inflammatory IL-6 respect to CMC and a significant secretion of Collagen Type I. CONCLUSIONS: PCL7-2i membranes showed biomimetic biomechanical properties and in-vitro biological properties similar to or even better than - in the case of anti-inflammatory behavior and collagen production - CMC, a commercially available product, suggesting potentially improved integration in the host tissue

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations

Nordic dietary patterns and cardiometabolic outcomes : a systematic review and meta-analysis of prospective cohort studies and randomised controlled trials

Funding Information: AZ is a part-time research associate at INQUIS Clinical Research (formerly Glycemic Index Laboratories), a contract research organisation, and a consultant for the Glycemic Index Foundation. AJG has received consulting fees from Solo GI Nutrition and an honorarium from the Soy Nutrition Institute. LC was a Mitacs Elevate postdoctoral fellow jointly funded by the Government of Canada and the Canadian Sugar Institute. She was previously employed as a casual clinical coordinator at INQUIS Clinical Research. TAK has received research support from the CIHR, the International Life Science Institute (ILSI) and the National Honey Board. He has been an invited speaker at the Calorie Control Council Annual Meeting for which he received an honorarium. EMC reports grants from the Natural Sciences and Engineering Research Council of Canada and the CIHR while this study was being conducted, has received research support from Lallemand Health Solutions and Ocean Spray, and has received consultant fees and speaker and travel support from Danone and Lallemand Health Solutions (all are outside this study). DR is director of Vuk Vrhovac University Clinic for Diabetes, Endocrinology and Metabolic Diseases at Merkur University Hospital, Zagreb, Croatia. He is the president of the Croatian Society for Diabetes and Metabolic Disorders of the Croatian Medical Association. He serves as an Executive Committee member of the Croatian Endocrine Society, Croatian Society of Obesity and Croatian Society for Endocrine Oncology. He was a board member and secretary of IDF Europe and is currently the chair of the IDF Young Leaders in Diabetes (YLD) Programme. He has served as an Executive Committee member of the Diabetes and Nutrition Study Group of the EASD and currently serves as an Executive Committee member of the Diabetes and Cardiovascular Disease Study Group of the EASD. He has served as principal investigator or co-investigator in clinical trials for AstraZeneca, Eli Lilly, MSD, Novo Nordisk, Sanofi Aventis, Solvay and Trophos. He has received travel support, speaker fees and honoraria for advisory board engagements and/or consulting fees from Abbott, Amgen, AstraZeneca, Bayer, Belupo, Boehringer Ingelheim, Eli Lilly, LifeScan – Johnson & Johnson, the International Sweeteners Association, Krka, Medtronic, Mediligo, Mylan, Novartis, Novo Nordisk, MSD, Pfizer, Pliva, Roche, Salvus, Sandoz, Solvay, Sanofi Aventis and Takeda. HK is Director of Clinical Research at the Physicians Committee for Responsible Medicine, a non-profit organisation that provides nutrition education and research. JS-S reports serving on the board of and receiving grant support through his institution from the International Nut and Dried Fruit Council (INC) and the Eroski Foundation. He reports serving on the Executive Committee of the Instituto Danone Spain. He reports receiving research support from the Instituto de Salud Carlos III, Spain; Ministerio de Educación y Ciencia, Spain; the Departament de Salut Pública de la Generalitat de Catalunya, Catalonia, Spain; the European Commission; the California Walnut Commission, USA; Patrimonio Comunal Olivarero, Spain; La Morella Nuts, Spain; and Borges, Spain. He reports receiving consulting fees or travel expenses from Danone, the California Walnut Commission, the Eroski Foundation, the Instituto Danone Spain, Nuts for Life, the Australian Nut Industry Council, Nestlé, Abbot and Font Vella y Lanjarón. He is on the Clinical Practice Guidelines Expert Committee of the EASD and served on the Scientific Committee of the Spanish Agency for Food Safety and Nutrition and the Spanish Federation of the Scientific Societies of Food, Nutrition and Dietetics. He is a member of the International Carbohydrate Quality Consortium (ICQC) and an Executive Board Member of the Diabetes and Nutrition Study Group of the EASD. CWCK has received grants or research support from the Advanced Food and Materials Network, Agriculture and Agri-Food Canada (AAFC), the Almond Board of California, Barilla, the CIHR, the Canola Council of Canada, the International Nut and Dried Fruit Council, the International Tree Nut Council Nutrition Research and Education Foundation, Loblaw Brands, the Peanut Institute, Pulse Canada and Unilever. He has received in-kind research support from the Almond Board of California, Barilla, the California Walnut Commission, Kellogg Canada, Loblaw Brands, Nutrartis, Quaker (PepsiCo), the Peanut Institute, Primo, Unico, Unilever, WhiteWave Foods/Danone. He has received travel support and/or honoraria from Barilla, the California Walnut Commission, the Canola Council of Canada, General Mills, the International Nut and Dried Fruit Council, the International Pasta Organization, Lantmannen, Loblaw Brands, the Nutrition Foundation of Italy, the Oldways Preservation Trust, Paramount Farms, the Peanut Institute, Pulse Canada, Sun-Maid, Tate & Lyle, Unilever and White Wave Foods/Danone. He has served on the scientific advisory board for the International Tree Nut Council, International Pasta Organisation, McCormick Science Institute and Oldways Preservation Trust. He is a founding member of the ICQC and an Executive Board Member of the Diabetes and Nutrition Study Group of the EASD, is on the Clinical Practice Guidelines Expert Committee for Nutrition Therapy of the EASD and is a Director of the Toronto 3D Knowledge Synthesis and Clinical Trials foundation. JLS has received research support from the Canadian Foundation for Innovation, the Ontario Research Fund, the Province of Ontario Ministry of Research, Innovation and Science, the CIHR, Diabetes Canada, the American Society for Nutrition (ASN), the International Nut and Dried Fruit Council Foundation, the National Honey Board (US Department of Agriculture [USDA] honey ‘Checkoff’ programme), the Institute for the Advancement of Food and Nutrition Sciences (IAFNS; formerly ILSI North America), Pulse Canada, the Quaker Oats Center of Excellence, the United Soybean Board (USDA soy ‘Checkoff’ programme), the Tate and Lyle Nutritional Research Fund at the University of Toronto, the Glycemic Control and Cardiovascular Disease in Type 2 Diabetes Fund at the University of Toronto (established by the Alberta Pulse Growers), the Plant Protein Fund at the University of Toronto (which has received contributions from IFF) and the Nutrition Trialists Fund at the University of Toronto (established by an inaugural donation from the Calorie Control Council). He has received food donations to support RCTs from the Almond Board of California, the California Walnut Commission, the Peanut Institute, Barilla, Unilever/Upfield, Unico/Primo, Loblaw Companies, Quaker, Kellogg Canada, WhiteWave Foods/Danone, Nutrartis and Dairy Farmers of Canada. He has received travel support, speaker fees and/or honoraria from the ASN, Danone, Dairy Farmers of Canada, FoodMinds, Nestlé, Abbott, General Mills, the Comité Européen des Fabricants de Sucre (CEFS), Nutrition Communications, the International Food Information Council (IFIC), the Calorie Control Council and the International Glutamate Technical Committee. He has or has had ad hoc consulting arrangements with Perkins Coie, Tate & Lyle, Phynova and INQUIS Clinical Research. He is a member of the European Fruit Juice Association Scientific Expert Panel and former member of the Soy Nutrition Institute Scientific Advisory Committee. He is on the Clinical Practice Guidelines Expert Committees of Diabetes Canada, the EASD, the Canadian Cardiovascular Society and Obesity Canada/Canadian Association of Bariatric Physicians and Surgeons. He serves or has served as an unpaid member of the Board of Trustees and an unpaid scientific advisor for the Food, Nutrition, and Safety Program (FNSP) and the Carbohydrates Committee of the IAFNS. He is a member of the ICQC, an Executive Board Member of the Diabetes and Nutrition Study Group of the EASD, and Director of the Toronto 3D Knowledge Synthesis and Clinical Trials foundation. His spouse is an employee of AB InBev. PM, EV, SBM, VC, US, UR, MU, A-MA, KH and IT declare that there are no relationships or activities that might bias, or be perceived to bias, their work. Funding Information: Open access funding provided by University of Eastern Finland (UEF) including Kuopio University Hospital. The Diabetes and Nutrition Study Group of the EASD commissioned this systematic review and meta-analysis and provided funding and logistical support for meetings as part of the development of the EASD clinical practice guidelines for nutrition therapy. This work was also supported by the Canadian Institutes of Health Research (CIHR; reference no. 129920) through the Canada-wide Human Nutrition Trialists’ Network (NTN). The Diet, Digestive tract, and Disease (3D) Centre, funded through the Canada Foundation for Innovation and the Ministry of Research and Innovation’s Ontario Research Fund, provided the infrastructure for the conduct of this work. PM was funded by a Connaught Fellowship, an Onassis Foundation Fellowship and a Peterborough KM Hunter Charitable Foundation Scholarship. AZ was funded by a Toronto3D Postdoctoral Fellowship Award and a Banting and Best Diabetes Centre (BBDC) Fellowship in Diabetes Care. AJG was funded by a Nora Martin Fellowship in Nutritional Sciences, the Banting & Best Diabetes Centre Tamarack Graduate Award in Diabetes Research, the Peterborough K. M. Hunter Charitable Foundation Graduate Award and an Ontario Graduate Scholarship. LC was funded by a Mitacs Elevate Postdoctoral Fellowship Award. TAK was funded by a Toronto 3D Postdoctoral Fellowship Award. EMC held the Lawson Family Chair in Microbiome Nutrition Research at the Lawson Centre for Child Nutrition, Temerty Faculty of Medicine, University of Toronto. JS-S is partially supported by the Catalan Institution for Research and Advanced Studies (ICREA) under the ICREA Acadèmia programme. JLS was funded by a PSI Graham Farquharson Knowledge Translation Fellowship, Canadian Diabetes Association Clinician Scientist Award, CIHR Institute of Nutrition, Metabolism and Diabetes (INMD)/Canadian Nutrition Society (CNS) New Investigator Partnership Prize and BBDC Sun Life Financial New Investigator Award. Publisher Copyright: © 2022, The Author(s).AIMS/HYPOTHESIS: Nordic dietary patterns that are high in healthy traditional Nordic foods may have a role in the prevention and management of diabetes. To inform the update of the EASD clinical practice guidelines for nutrition therapy, we conducted a systematic review and meta-analysis of Nordic dietary patterns and cardiometabolic outcomes. METHODS: We searched MEDLINE, EMBASE and The Cochrane Library from inception to 9 March 2021. We included prospective cohort studies and RCTs with a follow-up of ≥1 year and ≥3 weeks, respectively. Two independent reviewers extracted relevant data and assessed the risk of bias (Newcastle-Ottawa Scale and Cochrane risk of bias tool). The primary outcome was total CVD incidence in the prospective cohort studies and LDL-cholesterol in the RCTs. Secondary outcomes in the prospective cohort studies were CVD mortality, CHD incidence and mortality, stroke incidence and mortality, and type 2 diabetes incidence; in the RCTs, secondary outcomes were other established lipid targets (non-HDL-cholesterol, apolipoprotein B, HDL-cholesterol, triglycerides), markers of glycaemic control (HbA 1c, fasting glucose, fasting insulin), adiposity (body weight, BMI, waist circumference) and inflammation (C-reactive protein), and blood pressure (systolic and diastolic blood pressure). The Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach was used to assess the certainty of the evidence. RESULTS: We included 15 unique prospective cohort studies (n=1,057,176, with 41,708 cardiovascular events and 13,121 diabetes cases) of people with diabetes for the assessment of cardiovascular outcomes or people without diabetes for the assessment of diabetes incidence, and six RCTs (n=717) in people with one or more risk factor for diabetes. In the prospective cohort studies, higher adherence to Nordic dietary patterns was associated with 'small important' reductions in the primary outcome, total CVD incidence (RR for highest vs lowest adherence: 0.93 [95% CI 0.88, 0.99], p=0.01; substantial heterogeneity: I 2=88%, p Q<0.001), and similar or greater reductions in the secondary outcomes of CVD mortality and incidence of CHD, stroke and type 2 diabetes (p<0.05). Inverse dose-response gradients were seen for total CVD incidence, CVD mortality and incidence of CHD, stroke and type 2 diabetes (p<0.05). No studies assessed CHD or stroke mortality. In the RCTs, there were small important reductions in LDL-cholesterol (mean difference [MD] -0.26 mmol/l [95% CI -0.52, -0.00], p MD=0.05; substantial heterogeneity: I 2=89%, p Q<0.01), and 'small important' or greater reductions in the secondary outcomes of non-HDL-cholesterol, apolipoprotein B, insulin, body weight, BMI and systolic blood pressure (p<0.05). For the other outcomes there were 'trivial' reductions or no effect. The certainty of the evidence was low for total CVD incidence and LDL-cholesterol; moderate to high for CVD mortality, established lipid targets, adiposity markers, glycaemic control, blood pressure and inflammation; and low for all other outcomes, with evidence being downgraded mainly because of imprecision and inconsistency. CONCLUSIONS/INTERPRETATION: Adherence to Nordic dietary patterns is associated with generally small important reductions in the risk of major CVD outcomes and diabetes, which are supported by similar reductions in LDL-cholesterol and other intermediate cardiometabolic risk factors. The available evidence provides a generally good indication of the likely benefits of Nordic dietary patterns in people with or at risk for diabetes. REGISTRATION: ClinicalTrials.gov NCT04094194. FUNDING: Diabetes and Nutrition Study Group of the EASD Clinical Practice.Peer reviewe

The role of immune suppression in COVID-19 hospitalization: clinical and epidemiological trends over three years of SARS-CoV-2 epidemic

Specific immune suppression types have been associated with a greater risk of severe COVID-19 disease and death. We analyzed data from patients &gt;17 years that were hospitalized for COVID-19 at the “Fondazione IRCCS Ca′ Granda Ospedale Maggiore Policlinico” in Milan (Lombardy, Northern Italy). The study included 1727 SARS-CoV-2-positive patients (1,131 males, median age of 65 years) hospitalized between February 2020 and November 2022. Of these, 321 (18.6%, CI: 16.8–20.4%) had at least one condition defining immune suppression. Immune suppressed subjects were more likely to have other co-morbidities (80.4% vs. 69.8%, p &lt; 0.001) and be vaccinated (37% vs. 12.7%, p &lt; 0.001). We evaluated the contribution of immune suppression to hospitalization during the various stages of the epidemic and investigated whether immune suppression contributed to severe outcomes and death, also considering the vaccination status of the patients. The proportion of immune suppressed patients among all hospitalizations (initially stable at &lt;20%) started to increase around December 2021, and remained high (30–50%). This change coincided with an increase in the proportions of older patients and patients with co-morbidities and with a decrease in the proportion of patients with severe outcomes. Vaccinated patients showed a lower proportion of severe outcomes; among non-vaccinated patients, severe outcomes were more common in immune suppressed individuals. Immune suppression was a significant predictor of severe outcomes, after adjusting for age, sex, co-morbidities, period of hospitalization, and vaccination status (OR: 1.64; 95% CI: 1.23–2.19), while vaccination was a protective factor (OR: 0.31; 95% IC: 0.20–0.47). However, after November 2021, differences in disease outcomes between vaccinated and non-vaccinated groups (for both immune suppressed and immune competent subjects) disappeared. Since December 2021, the spread of the less virulent Omicron variant and an overall higher level of induced and/or natural immunity likely contributed to the observed shift in hospitalized patient characteristics. Nonetheless, vaccination against SARS-CoV-2, likely in combination with naturally acquired immunity, effectively reduced severe outcomes in both immune competent (73.9% vs. 48.2%, p &lt; 0.001) and immune suppressed (66.4% vs. 35.2%, p &lt; 0.001) patients, confirming previous observations about the value of the vaccine in preventing serious disease