Sleep duration and subclinical atherosclerosis: The Aragon Workers' Health Study

Background and aims: Few studies have evaluated the association of sleep duration with subclinical atherosclerosis, and with heterogeneous findings. We evaluated the association of sleep duration with the presence of coronary, carotid, and femoral subclinical atherosclerosis in healthy middle-age men with low prevalence of clinical comorbidities. Methods: We performed a cross-sectional analysis of 1968 men, 40–60 years of age, participating in the Aragon Workers' Health Study (AWHS). Duration of sleep during a typical work week was assessed by questionnaire. Coronary artery calcium scores (CACS) was assessed by computed tomography and the presence of carotid plaque and femoral plaque by ultrasound. Results: In fully adjusted models, the odds ratios (95% CI) for CACS >0 comparing sleep durations of =5, 6, and =8 h with 7 h were 1.34 (0.98–1.85), 1.35 (1.08–1.69) and 1.21 (0.90–1.62), respectively (p = 0.04). A similar U-shaped association was observed for CACS =100 and for CACS. The corresponding odds ratios for the presence of at least one carotid plaque were =5, 6, and =8 h with 7 h were 1.23 (0.88–1.72), 1.09 (0.86–1.38), and 0.86 (0.63–1.17), respectively (p = 0.31), and for the presence of at least one femoral plaque were 1.25 (0.87–1.80), 1.19 (0.93–1.51) and 1.17 (0.86–1.61), respectively (p = 0.39). Conclusions: Middle-aged men reporting 7 h of sleep duration had the lowest prevalence of subclinical coronary atherosclerosis as assessed by CACs. Our results support that men with very short or very long sleep durations are at increased risk of atherosclerosis

Laponite as carrier for controlled in vitro delivery of dexamethasone in vitreous humor models

Laponite clay is able to retain dexamethasone by simple physisorption, presumably accomplished by hydrogen bonding formation and/or complexation with sodium counterions, as shown by solid state NMR. The physisorption can be somehow modulated by changing the solvent in the adsorption process. This simple system is able to deliver dexamethasone in a controlled manner to solutions used as models for vitreous humor. The proven biocompatibility of laponite as well as its transparency in the gel state, together with the simplicity of the preparation method, makes this system suitable for future in vivo tests of ophthalmic treatment.This study was supported by the Instituto de Salud Carlos III (project PI12/02285) and authors would like to acknowledge the financial support received from Diputación General de Aragón (E11 Group co-financed by the European Regional Development Funds).Peer Reviewe

Monitoring new long-lasting intravitreal formulation for glaucoma with vitreous images using optical coherence tomography

Intravitreal injection is the gold standard therapeutic option for posterior segment patholo-gies, and long-lasting release is necessary to avoid reinjections. There is no effective intravitreal treatment for glaucoma or other optic neuropathies in daily practice, nor is there a non-invasive method to monitor drug levels in the vitreous. Here we show that a glaucoma treatment combining a hypotensive and neuroprotective intravitreal formulation (IF) of brimonidine–Laponite (BRI/LAP) can be monitored non-invasively using vitreoretinal interface imaging captured with optical coherence tomography (OCT) over 24 weeks of follow-up. Qualitative and quantitative characterisation was achieved by analysing the changes in vitreous (VIT) signal intensity, expressed as a ratio of retinal pigment epithelium (RPE) intensity. Vitreous hyperreflective aggregates mixed in the vitreous and tended to settle on the retinal surface. Relative intensity and aggregate size progressively decreased over 24 weeks in treated rat eyes as the BRI/LAP IF degraded. VIT/RPE relative intensity and total aggregate area correlated with brimonidine levels measured in the eye. The OCT-derived VIT/RPE relative intensity may be a useful and objective marker for non-invasive monitoring of BRI/LAP IF

RASSF1A–LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2

Genomic instability is a key hallmark of cancer leading to tumour heterogeneity and therapeutic resistance. ​BRCA2 has a fundamental role in error-free DNA repair but also sustains genome integrity by promoting ​RAD51 nucleofilament formation at stalled replication forks. ​CDK2 phosphorylates ​BRCA2 (pS3291-​BRCA2) to limit stabilizing contacts with polymerized ​RAD51; however, how replication stress modulates ​CDK2 activity and whether loss of pS3291-​BRCA2 regulation results in genomic instability of tumours are not known. Here we demonstrate that the Hippo pathway kinase ​LATS1 interacts with ​CDK2 in response to genotoxic stress to constrain pS3291-​BRCA2 and support ​RAD51 nucleofilaments, thereby maintaining genomic fidelity during replication stalling. We also show that ​LATS1 forms part of an ​ATR-mediated response to replication stress that requires the tumour suppressor ​RASSF1A. Importantly, perturbation of the ​ATR–​RASSF1A–​LATS1 signalling axis leads to genomic defects associated with loss of ​BRCA2 function and contributes to genomic instability and ‘BRCA-ness’ in lung cancers

Comparison of Proliferation and Genomic Instability Responses to WRN Silencing in Hematopoietic HL60 and TK6 Cells

BACKGROUND: Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges. CONCLUSIONS/SIGNIFICANCE: Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency

Controlled release for formulations

In this sense, we herein describe, for the first time, the first report of immobilization of a non-ionic drug (dexamethasone) on a Laponite clay, wherein the clay is not used as a component of a polyurethane composite.Peer reviewedUniversidad de Zaragoza, Fundación Instituto de Investigación Sanitaria Aragón (IIS Aragón), Consejo Superior de Investigaciones Científicas (CSIC)A1 Solicitud de patente con informe sobre el estado de la técnic

Laponite clay as a carrier for intravitreal dexamethasone release

Resumen del trabajo presentado a la XVI International Clay Conference (ICC), celebrada en Granada (España) del 17 al 21 de julio de 2017.Intravitreal injection is considered an effective approach for treatment of posterior segment ocular diseases, as it delivers the therapeutic agent directly into the eye, but it presents some potential risks associated to the need for repeated injections. Alternative drug delivery systems are being developed to overcome this kind of limitations by reducing the frequency of injections, for example using biodegradable polymeric implants for slow release of dexamethasone (an anti-inflammatory steroid), but this methodology requires a surgical intervention to implant the material. Clays have been recently envisaged as carriers for drug delivery, but they had never been used for ophthalmic treatment. Laponite (LAP) is a white synthetic smectite clay, able to form transparent colloidal dispersions in water. We have demonstrated that dexamethasone (DEX), in spite of being a neutral molecule, is retained on laponite through weak interactions, mainly hydrogen bonds, as shown by solid state NMR. In vitro studies have shown that dexamethasone is released below its solubility in saline solution or in a model of vitreous humor, under equilibrium conditions. The in vivo ocular biocompatibility of laponite has been assessed by eye examination (slit lamp and indirect ophthalmoscopy) after intravitreal administration to rabbits. The clay did not elicit any inflammatory response even if the clay could be observed floating into the vitreous body as a transparent gel from day 1 through week 14 postadministration. The small amount of eyes with cataract were probably due to a traumatic effect associated with the injection technique because the histological examination did not show any change in eye's lens structure. Thus, laponite could be considered as a vehicle for potential clinical use in ocular drug administration due to its proved ocular biocompatibility and its transparency in the gel state. Moreover, Mg content in vitreous humor showed a decrease along the 14 weeks. About 33% of total initial LAP-dose administered could be detected in vitreous humor for up to 14 weeks postadministration, indicating some kind of degradation and/or elimination of the clay through an unknown mechanism. Intravitreal injections of 100 μL dexamethasone/laponite dispersion (10 mg solid/mL, 1:10 DEX/LAP w/w) were performed in albino rabbits (New Zealand) and DEX concentrations in vitreous humor were monitored over 24 weeks. Vitreous dexamethasone levels remained relatively stable from 7th day postadministration, and showed an elimination rate significantly lower than that found after intravitreal injection of dexamethasone in solution (1 mg/mL) (CL= 0.49 vs 315.29 g/day). The administration of DEX loading to laponite can lengthen the half-life of the drug (T½-el= 132.75 vs 0.13 days) and enhance its clinical uses.Peer reviewe

Dexamethasone delivery to the ocular posterior segment by sustained-release Laponite formulation

This paper presents a novel nanoformulation for sustained-release delivery of dexamethasone (DEX) to the ocular posterior segment using a Laponite (LAP) carrier—DEX/LAP 1:10 w w−1 formulation; 10 mg ml−1. In vivo ocular feasibility and pharmacokinetics after intravitreal (IV) and suprachoroidal (SC) administration in rabbit eyes are compared against IV administration of a DEX solution (1 mg ml−1). Thirty rabbit eyes were injected with the DEX/LAP formulation (15 suprachoroid/15 intravitreous). Ophthalmological signs were monitored at day 1 and at weeks 1–4–12–24 post-administration. Three eyes per sample time point were used to quantify DEX concentration using high-performance liquid chromatography-mass spectrometry. The ocular tissues' pharmacokinetic parameters (lens, vitreous humour, choroid-retina unit and sclera) were studied. DEX/LAP was well tolerated under both administration methods. Peak intraocular DEX levels from the DEX/LAP were detected in the vitreous humour after both deliveries soon after administration. The vitreous area under the curve was significantly greater after both DEX/LAP deliveries (IV: 205 968.47; SC: 11 442.22 ng g−1 d−1) than after IV administration of the DEX solution (317.17 ng g−1 d−1). Intravitreal DEX/LAP delivery extended higher vitreous DEX levels up to week 24 (466.32 ± 311.15 ng g−1). With SC delivery, DEX levels were detectable in the choroid-retina unit (12.04 ± 20.85 ng g−1) and sclera (25.46 ± 44.09 ng g−1) up to week 24. This study demonstrated the intraocular feasibility of both SC and IV administration of the DEX/LAP formulation. The LAP increased the intraocular retention time of DEX when compared with conventional solutions. DEX/LAP could be considered a biocompatible and useful sustained-release formulation for treating posterior-pole eye diseases.This paper was supported by the Carlos III Health Institute (Programme Grant PI12/02285), by Rio Hortega Research Grant M17/00213, PI17/01726, PI17/01946 (Carlos III Health Institute), by MAT2017-83858-C2-2 MINECO/AEI/FEDER, EU, and by the Regional Government of Aragon (E37_17R group, co-financed under FEDER 2014–2020 'Construyendo Europa desde Aragón').Peer reviewe