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SummaryThe yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. Here, we report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Unexpectedly, IN1 binding associates with insertion of subunit C11 C-terminal Zn-ribbon into the Pol III funnel, which provides atomic evidence for a two-metal mechanism during RNA cleavage. Moreover, unstructured regions of subunits C53 and C37 reorganize close to C11, likely explaining the connection between the C37/C53 heterodimer and C11 during transcription reinitiation. Our results suggest that IN1 binding induces a Pol III configuration that favors chromatin residence, thus improving the likelihood of Ty1 integration.

Senescent cells accumulate with obesity in the white adipose tissue of mice and humans. These senescent cells enhance the pro-inflammatory environment that, with time, contributes to the onset of glucose intolerance and type 2 diabetes. Glucose intolerance in mouse models of obesity has been successfully reversed by the elimination of senescent cells with the senolytic compounds navitoclax or the combination of dasatinib and quercetin (D/Q). In this work, we generated obese mice by high-fat diet feeding, and treated them with five consecutive cycles of navitoclax or D/Q during 16 weeks. We observed an efficient reduction in the white adipose tissue of the senescence markers senescence-associated β-galactosidase activity, Cdkn2a-p16 and Cdkn2a-p19 at the end of the 5 cycles. Mice treated with both navitoclax and D/Q showed an improvement of their insulin sensitivity and glucose tolerance during a short period of time (cycles 3 and 4), that disappeared at the fifth cycle. Also, these mice tended to increase the expression at their adipose tissue of the adipogenic genes Pparg and, Cebpa, as well as their plasma adiponectin levels. Together, our work shows that two different senolytic treatments, acting through independent pathways, are transiently effective in the treatment of obesity-induced metabolic disorders.

Background Non-alcoholic fatty liver disease (NAFLD), a condition that leads to fibrosis, is caused by intake of very high-fat diets (HFDs). However, while the negative impact on the liver of these diets has been an issue of interest, systematic research on the effect of HFDs are lacking. Objective To characterize the overall impact of HFDs on both molecular and morphological signs of liver remodeling. Methods A study was conducted on male C57BL/6J mice to assess the effect of 4- and 8-week HFDs (60% kcal from fat) on (i) liver steatosis and fibrosis, and (ii) expression of factors involved in inflammation and angiogenesis. Results After an 8-week HFD, vascular endothelial growth factor type-2 receptor (VEGF-R2) and fatty acid translocase/trombospondin-1 receptor (CD36) were overexpressed in liver tissue of mice given HFDs. These changes suggest impaired liver angiogenesis and occurred together with (i) increased GPR78-BiP and EIF2α phosphorylation, suggesting endoplasmic reticulum stress, (ii) induction of Col1a1 gene expression, a marker of fibrosis, and (iii) increased CD31 immunolabeling, consistent with active angiogenesis and fibrosis. Conclusion Our data show that very HFDs promote a rapid inflammatory response, as well as deregulation of angiogenesis, both consistent with development of liver fibrosis.

Fasting exerts beneficial effects in mice and humans, including protection from chemotherapy toxicity. To explore the involved mechanisms, we collect blood from humans and mice before and after 36 or 24 hours of fasting, respectively, and measure lipid composition of erythrocyte membranes, circulating micro RNAs (miRNAs), and RNA expression at peripheral blood mononuclear cells (PBMCs). Fasting coordinately affects the proportion of polyunsaturated versus saturated and monounsaturated fatty acids at the erythrocyte membrane; and reduces the expression of insulin signaling-related genes in PBMCs. When fasted for 24 hours before and 24 hours after administration of oxaliplatin or doxorubicin, mice show a strong protection from toxicity in several tissues. Erythrocyte membrane lipids and PBMC gene expression define two separate groups of individuals that accurately predict a differential protection from chemotherapy toxicity, with important clinical implications. Our results reveal a mechanism of fasting associated with lipid homeostasis, and provide biomarkers of fasting to predict fasting-mediated protection from chemotherapy toxicity. General: We thank Prof. Jose Maria. Ordovas for his kind suggestions; nutritionists Helena Marcos-Pasero, Elena Aguilar-Aguilar and Isabel Espinosa-Salinas for their help with volunteers management; Rosa Serrano for her help with animal experiments; Susana Molina for her advice with PBMC isolation; Luisa Mariscal, Domingo Fernandez, Lola Martinez, Diego Megias, Patricia Gonzalez, Fernando Pelaez, Anabel Sanz, Carolina Pola, Celia de la Calle, Ana Ortega, Ana Sagrera, Jose Miguel Frade, Elena Lopez-Guadamillas, Maribel Munoz, Susana Llanos, Andres Fernandez, Aranzazu Sierra, Andres Lopez, Noemi Haro and Ildefonso Rodriguez for their excellent technical and scientific support. Work at the laboratory of P.J.F.M. is funded by the Ramon Areces Foundation, (CIVP18A3891), Asociacion Espanola contra el Cancer-AECC (SIRTBIO-LABAE18008FERN), a Ramon y Cajal Award from the Spanish Ministry of Science, Innovation and Universities (MICINN) (RYC-2017-22335), RETOS projects Program of MICINN (SAF2017-85766-R) and the Portuguese Foundation for Science and Technology (FCT-MCTES, SFRH/BD/124022/2016). Work at the laboratory of ARM was funded by the MICINN (PID2019-110183RB-C21), Regional Government of Community of Madrid (P2018/BAA-4343-ALIBIRD2020-CM) and the Ramon Areces Foundation. Work at the laboratory of A.D.R. Funded by the Comunidad de Madrid-Talento Grant 2018-T1/BMD-11966 and the MICINN PID-2019-106893RA-100. Work at the laboratory of L.D. is funded by projects from the Health Research Fund (ISCIII FIS PI14/01374 and FISPI17/00508) and from a Manuel de Oya research fellowship from the Beer and Health Foundation. Work at the laboratory of A.E. is funded by a Ramon y Cajal Award from MICINN (RYC-2013-13546) and RETOS projects Program of the MICINN, co-funded by the European Regional Development Fund (ERDF) (SAF2015-67538-R). Work in the laboratory of M.S. was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Council (ERC-2014-AdG/669622), and the "laCaixa" Foundation. Sí

To evaluate the anticholinergic burden on discharge of patients treated for constipation in an emergency department (ED) and to assess the effect on emergency revisiting within 30 days.Observational retrospective cohort study. We collected cases with a discharge diagnosis of constipation after ED treatment between September 2018 and June 2019 and recorded information on all drugs taken and the anticholinergic burden of treatment. A revisit to the ED within 30 days was the primary outcome.We included 104 patients. A high anticholinergic burden of treatment was identified in 47 (56.6%), an intermediate burden in 30 (36.1%), and a low burden in 6 (7.2%). Twenty-nine (27.9%) patients revisited the ED within 30 days of discharge. An intermediate anticholinergic burden (23 patients [31.1%] vs 4 [13.3%]; P = .061) and high burden (19 [40.4%] vs 8 [14.1%]; P = .002] was associated with revisiting within 30 days in the univariate analysis. On multivariate analysis, a high anticholinergic burden was independently associated with a higher rate of revisiting than a low burden: adjusted odds ratio (aOR), 4.21; 95% CI, 1.07-16.5; P = .039. An intermediate load was not associated with more revisits, however: aOR, 1.27; 95% CI, 0.25-6.41; P = .776. Prescription of long-term treatment with laxatives on discharge did not reduce revisiting withing 30-days in the group with a high anticholinergic load (OR, 0.86; 95% CI, 0.48-3.27; P = .526), but it did have an effect in patients an intermediate burden (OR, 0.13; 95% CI, 0.02-0.99; P = .049).The prescription of drugs leading to a high anticholinergic burden was a factor associated with ED revisits within 30 days in patients treated for constipation.Evaluar la frecuencia e impacto de la carga anticolinérgica del tratamiento en la reconsulta a los 30 días en los pacientes atendidos por estreñimiento en un servicio de urgencias (SU).Estudio observacional de cohortes retrospectivo. Se incluyeron por oportunidad pacientes que fueron dados de alta con diagnóstico de estreñimiento desde un SU entre septiembre 2018 y junio 2019. Se recogieron los fármacos y su carga anticolinérgica. La variable de resultado fue la reconsulta por cualquier causa a los 30 días.Se incluyeron 104 pacientes, 47 (56,6%) se clasificaron como tratamiento con alta carga colinérgica, 30 (36,1%) intermedia y 6 (7,2%) baja. Veintinueve (27,9%) pacientes sufrieron una reconsulta a urgencias en los primeros 30 días tras el alta. Los pacientes con fármacos con una carga anticolinérgica alta tuvieron una mayor frecuencia de reconsultas a 30 días [19/47 (40,4%) vs 8/57 (14,1%); p = 0,002]. Tras el análisis multivarible, en comparación con aquellos con tratamiento con baja carga anticolinérgica, el tener una alta carga (ORa = 4,21; IC 95% 1,07-16,5; p = 0,039), pero no intermedia (ORa = 1,27; IC 95% 0,25-6,41; p = 0,776), se asoció de forma independiente con una mayor reconsulta a los 30 días. La prescripción de laxantes crónicos al alta no redujo la reconsulta a 30 días en el grupo con alta carga anticolinérgica (OR = 0,86; IC 95% 0,48-3,27; p = 0,526), pero sí en aquellos con carga intermedia (OR = 0,13; IC 95% 0,02-0,99; p = 0,049).La prescripción de fármacos con alta carga anticolinérgica fue un factor asociado con reconsulta a los 30 días en los pacientes atendidos por estreñimiento en urgencias.

Consumption of high-fat diets (HFD) has been associated with neuronal plasticity deficits and cognitive disorders linked to the alteration of glutamatergic disorders in the hippocampus. As young individuals are especially vulnerable to the effects of nutrients and xenobiotics on cognition, we studied the effect of chronic consumption of saturated (SOLF) and unsaturated oil-enriched foods (UOLF) on: i) spatial memory; ii) hippocampal synaptic transmission and plasticity; and iii) gene expression of glutamatergic receptors and hormone receptors in the hippocampus of adolescent and adult mice. Our results show that both SOLF and UOLF impair spatial short-term memory. Accordingly, hippocampal synaptic plasticity mechanisms underlying memory, and gene expression of NMDA receptor subunits are modulated by both diets. On the other hand, PPARγ gene expression is specifically down-regulated in adolescent SOLF individuals and up-regulated in adult UOLF mice.

Aim: Cholecystokinin (CCK) participates in the storage of dietary triglycerides in white adipose tissue (WAT). Our goal was to characterize, both in subcutaneous (Sc-WAT) and visceral WAT (Vis-WAT), the functional expression of the two known CCK receptors, CCK-1 (CCK-1R) and CCK-2 (CCK-2R), as well as of CCK. Main methods: Gene and protein expression was assessed in different cell types of rat and human WAT by means of RT-PCR and western-blot, respectively. The functionality of CCK-Rs was tested by quantifying protein kinase B (Akt) phosphorylation after treatment of pre-adipocytes with the bioactive fragment of CCK, CCK-8. The CCK receptor subtype involved in Akt phosphorylation was investigated by using selective CCK-1R (SR-27,897) and CCK-2R antagonists (L-365,260). Key findings: In rats, CCK-1R (Cckar) and CCK-2R (Cckbr) gene expression was detected in the two types of WAT analyzed as well as in isolated adipocytes, mesenchymal stem cells and pre-adipocytes. CCK-1R and CCK-2R proteins were identified in adipocytes and, to a minor extent, in pre-adipocytes. In addition, CCK-2R were detected in subcutaneous mesenchymal stem cells. Gene expression of the CCK precursor preproCCK as well as CCK immunoreactivity were also found in Sc-WAT and Vis-WAT. In human WAT, CCK gene expression as well as CCK-2Rs and CCK were also identified. CCK-8 evoked Akt phosphorylation in rat pre-adipocytes, and this effect was antagonized by SR-27,897 and L-365,260. Significance: Our data show that both human and rat WAT express a complete CCK system, and suggest that CCK may have an autocrine/paracrine role in regulating adipose tissue biology.

The incorporation of plasma triglyceride (TG) fatty acids to white adipose tissue (WAT) depends on lipoprotein lipase (LPL), which is regulated by angiopoietin-like protein-4 (ANGPTL-4), an unfolding molecular chaperone that converts active LPL dimers into inactive monomers. The production of ANGPTL-4 is promoted by fasting and repressed by feeding. We hypothesized that the postprandial hormone cholecystokinin (CCK) facilitates the storage of dietary TG fatty acids in WAT by regulating the activity of the LPL/ANGPTL-4 axis and that it does so by acting directly on CCK receptors in adipocytes. We report that administration of CCK-8 (a bioactive fragment of CCK) to rats: (i) reduces plasma ANGTPL-4 levels; (ii) repressesAngptl-4expression in WAT and (iii) simultaneously enhances LPL activity in this tissue without inducingLplexpression.In vivoCCK-8 effects are specifically antagonized by the CCK-2 receptor (CCK-2R) antagonist, L-365,260. Moreover, CCK-8 downregulatesAngptl-4expression in wild-type pre-adipocytes, an effect that is not observed in engineered pre-adipocytes lacking CCK-2R. These effects have functional consequences as CCK-8 was found to promote the uptake of dietary fatty acids by WAT, as demonstrated by means of proton nuclear magnetic resonance (1H-NMR). The efficacy of acute CCK-8 administration was not reduced after chronic CCK-8 treatment. Moreover, the effects of CCK-8 on WAT were not associated to the increase of circulating insulin. Our results show that cholecystokinin promotes lipid storage in WAT by acting on adipocyte CCK-2R, suggesting a pivotal role for CCK in TG homeostasis.

Neuroendocrine tumors (NET) comprise a rare and heterogeneous family of tumors arising from cells throughout the diffuse endocrine system. Carcinoid syndrome, a common gastrointestinal and lung NET complication, is mainly caused by serotonin release into the bloodstream, promoting diarrhea, flushing and carcinoid heat disease. By inhibiting the tryptophan hydroxylase, telotristat ethyl diminishes the synthesis of serotonin and improves carcinoid-related bowel movements. However, to our knowledge, there is no data showing the potential activity of telotristat ethyl as anti-tumoral agent and not only as a symptom-reliever . The purpose of this study is to analyze possible anti-tumoral effects of telotristat ethyl in NETs. For this, we used the pancreatic NET cell lines BON-1 and QGP1, and the gastrointestinal cell line HROC57. We treated them with telotristat ethyl as a single agent or in combination with common NET therapies, such as somatostatin analogues, mTOR inhibitors, standard chemotherapy and tyrosine kinase inhibitors. We analyzed the effects of these treatments on cellular viability, apoptosis markers, cell cycle and signaling pathways. Telotristal ethyl as single agent induced apoptosis measured by MTT in all NET cell lines after 72 hours of treatment, with IC50 in the µM range. Telotristat ethyl maintained this apoptotic effect even after serotonin addition. Surprisingly, neither the somatostatin analog octreotide nor the standard NET chemotherapy capecitabin/temozolomide induced apoptosis in BON-1, QGP-1 nor HROC57, suggesting that these cell lines are somatostatin analog- and chemotherapy-resistant. mTOR inhibition by everolimus treatment in NET cell lines induced apoptosis as monotherapy with IC50 in the µM range. Telotristat ethyl combination with everolimus produced a strong synergetic effect, dramatically decreasing cell viability in all NET cell lines. Mechanistically, combination of telotristat ethyl with everolimus promoted increasing levels of the apoptosis marker Anexin V with no major effects on cell cycle. mTOR inhibition by everolimus, alone and in combination with telotristat ethyl, decreased phosphorylated levels of the mTOR pathway readout protein S6 and elevated phosphorylated p42/44 MAPK levels, with no clear effects of telotristat ethyl. Our results show that telotristat ethyl has the capacity to inhibit pancreatic and GI NET cells tumor growth as a single agent and seems synergistic when combined with mTOR inhibitors but not with antiangiogenics or chemotherapy. Citation Format: Arantzazu Sierra Ramirez, Marinela Méndez, Adrián Plaza, Andrés Pastor, Javier Molina Cerrillo, Pablo José Fernández Marcos, Teresa Alonso Gordoa, Enrique Grande Pulido. Antitumoral effect of telotristat ethyl as a single agent in neuroendocrine tumor cell lines and potential synergies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P024.

The aim of this study was to indentify the involvement of leptin receptors (LepR) in astrocytes in hippocampal synaptic transmission and plasticity and metabolism. To this end we used a genetic mouse model (GFAP-LepR(-/-)) of specific LepR ablation in GFAP positive cells and recorded excitatory postsynaptic potentials (fEPSPs) within the CA1 area. Glutamate (Glu) uptake and the expression of Glu transporters (EEAT3, GLT-1 and GLAST) and enzymes involved in Glu metabolism (glutamine synthase, GABA decarboxylase 65 and 67) were quantified. Modifications in the expression of GFAP, the glucose transporter (GLUT)-1, and the monocarboxylate transporters MCT-2 and MCT-4, were also analyzed. The results show that depletion of LepR in GFAP positive cells reduced basal synaptic transmission within the CA1 area and impaired N-methyl-D-aspartate (NMDA)-evoked long-term depression (NMDA-LTD). Hippocampal slices from GFAP-LepR(-/-) mice displayed lower Glu uptake efficacy together with up-regulation of GLT-1, glutamine synthase, GFAP and GLUT-1. In conclusion, astrocyte LepRs are involved in the maintenance of Glu homeostasis and Glu neurotransmission within the hippocampus. Our findings support a role of hippocampal LepRs in synaptic plasticity, which could have an impact on memory and learning processes.This article is part of a Special Issue entitled: The Neuroscience of Energy Balance and Eating Behavior (C) 2019 IBRO.

Caloric restriction (CR) improves endothelial function through the upregulation of adenosine monophosphate-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Moreover, hydrogen peroxide (H

Caloric restriction (CR) improves endothelial function through the upregulation of adenosine monophosphateactivated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Moreover, hydrogen peroxide (H2O2) is upregulated in yeast subjected to CR. Our aim was to assess if mild short-term CR increases vascular H2O2 formation as a link with AMPK and eNOS activation. Twelve-week old Zucker obese (fa/fa) and control Zucker lean male rats were fed a standard chow either ad libitum (AL, n=10) or with a 20% CR (CR, n=10) for two weeks. CR significantly improved relaxation to ACh in fa/fa rats because of an enhanced endogenous production of H2O2 in aortic rings (H2O2 levels fa/faAL=0.5±0.05 nmol/mg vs. H2O2 levels fa/faCR=0.76±0.07 nmol/mg protein; p

Colorectal cancer has the second highest cancer-related mortality rate, with an estimated 881,000 deaths worldwide in 2018. The urgent need to reduce the incidence and mortality rate requires innovative strategies to improve prevention, early diagnosis, prognostic biomarkers, and treatment effectiveness. Caloric restriction (CR) is known as the most robust nutritional intervention that extends lifespan and delays the progression of age-related diseases, with remarkable results for cancer protection. Other forms of energy restriction, such as periodic fasting, intermittent fasting, or fasting-mimicking diets, with or without reduction of total calorie intake, recapitulate the effects of chronic CR and confer a wide range of beneficial effects towards health and survival, including anti-cancer properties. In this review, the known molecular, cellular, and organismal effects of energy restriction in oncology will be discussed. Energy-restriction-based strategies implemented in colorectal models and clinical trials will be also revised. While energy restriction constitutes a promising intervention for the prevention and treatment of several malignant neoplasms, further investigations are essential to dissect the interplay between fundamental aspects of energy intake, such as feeding patterns, fasting length, or diet composition, with all of them influencing health and disease or cancer effects. Currently, effectiveness, safety, and practicability of different forms of fasting to fight cancer, particularly colorectal cancer, should still be contemplated with caution.

Scope To identify the age-dependent effect of diets containing elevated amounts of either saturated or unsaturated fatty acids on cardiac steatosis in mice. Methods and results Five- and eight-week-old C57BL/6J mice cohorts are given free access to either a saturated or an unsaturated fatty-acid-enriched diet during 8 weeks. Body weight (BW) and food intake are monitored during this period. Cardiac lipid content, carnitine palmitoyltransferase-I (CPT-I) activity, and the amount of uncoupling proteins 2 and 3 (UCP2 and UCP3) are analyzed and correlated with blood leptin concentration. Leptin and PPARγ gene expression is quantified in white adipose tissue (WAT). Both diets have a similar effect on food intake, BW, and adiposity, independently of the age. Nevertheless, cardiac steatosis is specifically identified in adolescent mice consuming the saturated diet. These animals also display lower activity of cardiac CPT-I, a down-regulation of cardiac UCP2, together with lower concentration of plasma leptin. Accordingly, leptin gene expression is reduced in the visceral WAT. Conclusion Consumption of diets containing elevated amounts of saturated fat during adolescence and early adult life promotes cardiac steatosis in mice. An insufficient endocrine activity of WAT, in terms of leptin production, may account for such an effect.

Plants belonging to the genus Taraxacum are considered a nutritious food, being consumed raw or cooked. Additionally, these plants have long been used in folk medicine due to their choleretic, diuretic, antitumor, antioxidant, antiinflammatory, and hepatoprotective properties. This genus, with its complex taxonomy, includes several species that are difficult to distinguish. Its traditional use must be related not only to T. officinale F.H. Wigg., the most studied species, but also to others. The aim of this work is to compare five different common South European species of Taraxacum (T. obovatum (Willd.) DC., T. marginellum H. Lindb., T. hispanicum H. Lindb., T. lambinonii Soest and T. lacistrum Sahlin), in order to find differences between antioxidant and cytotoxic activities among them. Dissimilarities between species in LC/MS patterns, in in vitro and intracellular antioxidant activity and also in the cytotoxicity assay were found. T. marginellum was the most efficient extract reducing intracellular ROS levels although in in vitro assays, T. obovatum was the best free radical scavenger. A relevant cytotoxic effect was found in T. lacistrum extract over HeLa and HepG2 cell lines.