Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors

<p>Abstract</p> <p>Background</p> <p>The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as <it>ATM </it>(Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity.</p> <p>Results</p> <p>Using a replication-free assay in <it>Xenopus </it>extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells.</p> <p>Conclusions</p> <p>These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.</p

Height and body-mass index trajectories of school-aged children and adolescents from 1985 to 2019 in 200 countries and territories: a pooled analysis of 2181 population-based studies with 65 million participants

Summary Background Comparable global data on health and nutrition of school-aged children and adolescents are scarce. We aimed to estimate age trajectories and time trends in mean height and mean body-mass index (BMI), which measures weight gain beyond what is expected from height gain, for school-aged children and adolescents. Methods For this pooled analysis, we used a database of cardiometabolic risk factors collated by the Non-Communicable Disease Risk Factor Collaboration. We applied a Bayesian hierarchical model to estimate trends from 1985 to 2019 in mean height and mean BMI in 1-year age groups for ages 5–19 years. The model allowed for non-linear changes over time in mean height and mean BMI and for non-linear changes with age of children and adolescents, including periods of rapid growth during adolescence. Findings We pooled data from 2181 population-based studies, with measurements of height and weight in 65 million participants in 200 countries and territories. In 2019, we estimated a difference of 20 cm or higher in mean height of 19-year-old adolescents between countries with the tallest populations (the Netherlands, Montenegro, Estonia, and Bosnia and Herzegovina for boys; and the Netherlands, Montenegro, Denmark, and Iceland for girls) and those with the shortest populations (Timor-Leste, Laos, Solomon Islands, and Papua New Guinea for boys; and Guatemala, Bangladesh, Nepal, and Timor-Leste for girls). In the same year, the difference between the highest mean BMI (in Pacific island countries, Kuwait, Bahrain, The Bahamas, Chile, the USA, and New Zealand for both boys and girls and in South Africa for girls) and lowest mean BMI (in India, Bangladesh, Timor-Leste, Ethiopia, and Chad for boys and girls; and in Japan and Romania for girls) was approximately 9–10 kg/m2. In some countries, children aged 5 years started with healthier height or BMI than the global median and, in some cases, as healthy as the best performing countries, but they became progressively less healthy compared with their comparators as they grew older by not growing as tall (eg, boys in Austria and Barbados, and girls in Belgium and Puerto Rico) or gaining too much weight for their height (eg, girls and boys in Kuwait, Bahrain, Fiji, Jamaica, and Mexico; and girls in South Africa and New Zealand). In other countries, growing children overtook the height of their comparators (eg, Latvia, Czech Republic, Morocco, and Iran) or curbed their weight gain (eg, Italy, France, and Croatia) in late childhood and adolescence. When changes in both height and BMI were considered, girls in South Korea, Vietnam, Saudi Arabia, Turkey, and some central Asian countries (eg, Armenia and Azerbaijan), and boys in central and western Europe (eg, Portugal, Denmark, Poland, and Montenegro) had the healthiest changes in anthropometric status over the past 3·5 decades because, compared with children and adolescents in other countries, they had a much larger gain in height than they did in BMI. The unhealthiest changes—gaining too little height, too much weight for their height compared with children in other countries, or both—occurred in many countries in sub-Saharan Africa, New Zealand, and the USA for boys and girls; in Malaysia and some Pacific island nations for boys; and in Mexico for girls. Interpretation The height and BMI trajectories over age and time of school-aged children and adolescents are highly variable across countries, which indicates heterogeneous nutritional quality and lifelong health advantages and risks

Heterogeneous contributions of change in population distribution of body mass index to change in obesity and underweight NCD Risk Factor Collaboration (NCD-RisC)

From 1985 to 2016, the prevalence of underweight decreased, and that of obesity and severe obesity increased, in most regions, with significant variation in the magnitude of these changes across regions. We investigated how much change in mean body mass index (BMI) explains changes in the prevalence of underweight, obesity, and severe obesity in different regions using data from 2896 population-based studies with 187 million participants. Changes in the prevalence of underweight and total obesity, and to a lesser extent severe obesity, are largely driven by shifts in the distribution of BMI, with smaller contributions from changes in the shape of the distribution. In East and Southeast Asia and sub-Saharan Africa, the underweight tail of the BMI distribution was left behind as the distribution shifted. There is a need for policies that address all forms of malnutrition by making healthy foods accessible and affordable, while restricting unhealthy foods through fiscal and regulatory restrictions

Analyse transcriptionnelle des phases précoces de l'infection par le baculovirus AcMNPV et exploitation d'une banque d'ADN complémentaire issue de sa cellule-hôte, Sf9

S. frugiperda (Lepidoptera) is both a major pest of cultivated plants and the source of the Sf9 cell line, widely used for the production of recombinant proteins in the baculovirus expression system. In the first part of this work, we studied the transcriptional events occuring during the early steps of the viral infection. We first show that homologous regions (hrs) of the AcMNPV baculovirus contain a large number of binding sites for AP1/CREB transcription factors, that specifically bind proteins of the Sf9 host cell. Furthermore, in the infected context these sites are required for the transactivation mediated by the immediate-early viral factor IE1, that also binds hr sequences. This study shows for the first time the involvement of cellular factors in this mechanism of viral transactivation. However, this work has been impaired by the fact that little is known about the genes expressed in the Sf9 cell line. We thus decided to construct a cDNA library, that was used for different purposes: screening of the library using a probe previously identified in the lab allowed us to obtain the complete hsp90 gene sequence and to determine its main features. Besides this work, large-scale sequencing of the library led to the construction of an EST library, from which we identified almost all the ribosomal protein genes. Sequence analysis revealed several features that seemed to be restricted to insect or lepidoptera species, an unexpected result given the large conservation of these genes througout the evolution.Le lépidoptère Spodoptera frugiperda est un ravageur important des cultures mais aussi l'organisme source de la lignée cellulaire Sf9, utilisée pour la production de protéines recombinantes dans le système d'expression baculovirus/cellule d'insecte. Dans la première partie de ce travail, nous avons étudié la transcription chez le baculovirus AcMNPV aux temps précoces de l'infection. Nous montrons tout d'abord que les régions homologues (hrs) portées par le génome de ce virus contiennent de nombreux motifs de reconnaissance pour des facteurs de transcription de type AP1/CREB, et que ces sites fixent spécifiquement des protéines de la cellule-hôte, Sf9. Par ailleurs, dans le contexte de l'infection, ces sites sont nécessaires à la transactivation médiée par le facteur viral très précoce IE1 qui se fixe lui aussi sur les hrs. Cette étude montre pour la première fois l'implication de facteurs cellulaires dans ce mécanisme de transactivation virale. La progression de ces travaux a cependant été freinée par le peu de données disponibles sur les gènes exprimés par Sf9. Pour en faciliter l'accès, nous avons donc construit une banque d'ADNc, dont l'exploitation fait l'objet de la deuxième partie de ce mémoire. Son criblage a permis d'obtenir la séquence complète de l'ADN complémentaire du gène hsp90 chez S. frugiperda, et d'en déterminer les principales caractéristiques. Par ailleurs, le séquençage à grande échelle de la banque a conduit à la constitution d'une banque d'ESTs, permettant l'identification de la quasi-totalité des gènes de protéines ribosomales. L'analyse de leur séquence a révélé l'existence de particularités qui semblent restreintes aux insectes et aux lépidoptères, un résultat inattendu compte tenu de la grande conservation de ces gènes entre espèces

Analyse transcriptionnelle des phases précoces de l'infection par le baculovirus AcMNPV et exploitation d'une banque d'ADN complémentaire issue de la cellule hôte, Sf9

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Human Cytomegalovirus miR-UL112-3p Targets TLR2 and Modulates the TLR2/IRAK1/NFκB Signaling Pathway

<div><p>Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using <i>in silico</i> approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1β, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression.</p></div

TLR2 is down-regulated during HCMV infection.

<p>(A) TLR2 follows a biphasic expression pattern during HCMV AD169 infection of NHDF fibroblasts with early induction and late down-regulation. Numbers below the TLR2 blot represent quantification of the protein signal. IB, immunoblot. TRAM1 was used as loading control. (B) miR-UL112-3p accumulates at late time points during AD169 infection of NHDF cells. miR-UL112-3p copy number was determined by RT-PCR at various points during the infection of NHDF fibroblasts with HCMV AD169 (MOI: 3). (C) TLR2 is down-regulated late during TB40E infection of THP-1 monocytic cells. (D) miR-UL112-3p accumulates at late time points during TB40E infection of differentiated THP-1 cells (MOI: 3).</p

miR-UL112-3p inhibits TLR2-dependent activation of IRAK1.

<p>(A) Schematic of the upper section of the TLR2/NFκB pathway with known factors and post-translational modifications of IRAK4 and IRAK1 upon stimulation with TLR2 agonists. Grey ellipse, agonist; P, phosphorylation; K63, Lys63 poly-ubiquitin chain. (B) IRAK1 undergoes post-translational modifications upon stimulation with a TLR2/TLR1 agonist (PAM3CSK4, 100ng/ml) but not with a TLR4 agonist (LPS). TPA-differentiated THP-1 cells were treated as described in panel A. Unmodified and post-translationally modified forms of IRAK1 were detected by IB. (C) miR-UL112-3p inhibits FSL-1 induced IRAK1 post-translational modifications. TLR2, β-actin and IRAK1 unmodified and modified forms were detected by IB. Numbers below the IRAK1 blot represent quantification (in relative units) of the unmodified protein signal.</p

miR-UL112-3p inhibits TLR2-dependent activation of the NFκB pathway in THP1 cells.

<p>(A) Schematic of cell treatment. THP1 or NFκB luciferase reporter THP1 cells (THP1-NFκBRE-luc) were transfected with siRNA or miRNA mimics after being differentiated for 24hrs with TPA. Cells were stimulated 48hrs post-transfection with the TLR2/TLR6 agonist FSL-1 and harvested either 30 min or 5 hrs later for IB analysis or luciferase assay, respectively. (B) miR-UL112-3p inhibits NFκBRE-driven luciferase activity stimulated by the TLR2 agonist FSL-1 with similar efficacy as siTLR2. Each test was performed in quadruplicate. (C) miR-UL112-3p decreases TLR2 mRNA level (upper panel) and IL-1β, IL-6 and IL-8 mRNA expression stimulated by the TLR2 agonist FSL-1 (three lower panels) with similar efficacy as siTLR2. Each test was performed in duplicate. **, P-value <0.0001 (sample vs. NEG).</p

TLR2 3’UTR is targeted by HCMV miRNAs.

<p>(A) Dual luciferase reporter assay suggests that TLR2 3’UTR is targeted by miR-UL112-3p and miR-US25-2. Two independent pSICHECK2-TLR2 3’UTR clones were tested; stars indicate potential Renilla luciferase down-regulation. Putative and confirmed HCMV miRNAs are indicated in abbreviated form (see detailed list in legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004881#ppat.1004881.s001" target="_blank">S1 Fig</a>) (B) TLR2 3’UTR is down-regulated by miR-UL112-3p. HEK293 cells were transfected with pSICHECK2-TLR2 3’UTR and the indicated miRNA mimics alone or in combination. (C) Two potential miR-UL112-3p target site are present near the 5’end of TLR2 3’UTR (bold characters). Wobble G-U pairs are in italics; seed sequences are indicated. (D) Both target sites are required for full miR-UL112-3p down-regulation of TLR2. pSICHECK2 vectors containing mutations in target sites #1 and #2 (mut1 and mut2, respectively) were assayed by luciferase assay with the indicated miRNAs. (E) A miR-UL112-3p mimic down-regulates endogenous TLR2 in THP1 cells. TPA-differentiated THP1 cells left untransfected (Ø) or transfected as indicated were harvested 2 days post-transfection for IB analysis.</p