Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule "crystallization chaperone" 5'-O-tritylinosine (KIN59)

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5'-O-tritylinosine (previously designated KIN59) is a noncompetitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5'-O-tritylinosine on human TP. A cavity in which 5'-O-tritylinosine could fit was identified in the vicinity of the Gly405-WI419 loop at a distance of about 11 angstrom from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V-max /K-m of mutant enzyme similar to 50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5'-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP. (C) 2009 Elsevier Inc. All rights reserved

Biomass-modulated fire dynamics during the last glacial-interglacial transition at the central pyrenees (Spain)

Understanding long-term fire ecology is essential for current day interpretation of ecosystem fire responses. However palaeoecology of fire is still poorly understood, especially at high-altitude mountain environments, despite the fact that these are fire-sensitive ecosystems and their resilience might be affected by changing fire regimes. We reconstruct wildfire occurrence since the Lateglacial (14.7. cal. ka BP) to the Mid-Holocene (6. cal. ka BP) and investigate the climate-fuel-fire relationships in a sedimentary sequence located at the treeline in the Central Spanish Pyrenees. Pollen, macro- and micro-charcoal were analysed for the identification of fire events (FE) in order to detect vegetation post-fire response and to define biomass-fire interactions. mean fire intervals (mfi) reduced since the Lateglacial, peaking at 9-7.7. cal. ka BP while from 7.7 to 6. cal. ka BP no fire is recorded. We hypothesise that Early Holocene maximum summer insolation, as climate forcing, and mesophyte forest expansion, as a fuel-creating factor, were responsible for accelerating fire occurrence in the Central Pyrenees treeline. We also found that fire had long-lasting negative effects on most of the treeline plant communities and that forest contraction from 7.7. cal. ka BP is likely linked to the ecosystem's threshold response to high fire frequencies.This research has been funded by the projects DINAMO (CGL2009-07992) (funding EGPF — grant ref. BES-2010-038593 and MSC), DINAMO2 (CGL2012-33063), ARAFIRE (2012 GA LC 064), GRACCIE-CONSOLIDER (CSD2007-00067). GGR was funded by the Juan de la Cierva Program (grant ref. JCI2009-04345) and JAE-Doc CSIC Program, LLM was supported by a postdoctoral MINT fellowship funded by the Institute for the Environment (Brunel University), AMC is a Ramón y Cajal fellow (ref: RYC-2008-02431), APS holds a grant funded by the Aragon Government (ref. 17030G/5423/480072/14003) and JAE holds a grant funded by the Basque Country Government (BFI-2010-5)

Protein-ligand complex for structure-based design: impact on the affinity and antitumor activity of new tubulin ligands

Resumen del trabajo presentado en el XVIII Congreso de la Sociedad Española de Química Terapéutica, celebrado en Salamanca (España), del 23 al 26 de enero de 2018Microtubules, made of ¿ß¿tubulin heterodimers, are the key components of the cytoskeleton and play a crucial role in many cellular processes, such as cell motility, morphogenesis and mitosis.[1] Interference with microtubule dynamics induces cell cycle arrest during mitosis and triggers cell death. Compounds that interact with tubulin, especially those binding at the colchicine domain, have been deeply investigated as anticancer drugs due to their dual mechanism of action as antimitotics and as vascular disrupting agents.[2,3] Our research group has recently described a new family of colchicine¿domain binders, based on a cyclohexanedione skeleton, with potent antiproliferative activity against tumor and endothelial cells.[4] Moreover, to gain insight into the binding mode of these cyclohexanediones, we have determined the crystal structure of ¿ß¿tubulin in complex with our hit compound (TUB075). Based on this detailed information and by applying the affinity maps program cGRILL, a structurebased synthesis of new cyclohexanedione derivatives has been accomplished with the objective of improving their affinity for tubulin and their antitumor activity. Following this approach, we have obtained new compounds with potent antiproliferative activity against tumor and endothelial cells (IC50=8¿31 nM) and with the highest Kb value reported for compounds binding at the colchicine site in tubulin. Additional studies have shown that they arrest cell cycle at G2/M and disrupt a network of endothelial cells. Moreover they keep antiproliferative activity against cell lines overexpressing P¿gp, further supporting the potential of these compounds.The financial support of the Spanish MINECO (SAF2012‐39760‐C02‐01 and SAF 2015‐64629‐C2‐1‐R), Comunidad de Madrid (BIPEDD2; ref P2010/BMD‐2457) and the COST action CM1407 (to M J. P.P., S.L., M.O.S. and J.F.D.) is sincerely acknowledge

Norovirus outbreaks in long-term care facilities in Catalonia from 2017 to 2018

Norovirus is the leading cause of outbreaks of acute viral gastroenteritis. We carried out this study to investigate outbreaks in long-term care facilities reported in 2017 and 2018 in Catalonia (Spain). The characteristics of the centers, exposed persons and the genogroups responsible were analyzed. Viral loads were estimated. The attack rate (AR) of the outbreaks studied, and the rate ratio (RR) and the odds ratio (OR) and their 95% confidence intervals as measures of association were calculated. The mean cycle thresholds were compared using the t-test for independent means. We included 30 outbreaks (4631 exposed people). The global AR was 25.93%. The RR of residents vs. staff was 2.28 (95% CI 2.0-2.6). The RR between AR in residents with total or severe dependence vs. residents with moderate, low or no-dependence was 1.23 (95% CI 1.05-1.45). The AR were higher in smaller centers than in larger ones (38.47% vs. 19.25% and RR 2; 95% CI 1.82-2.2). GII was responsible for 70% of outbreaks. No association was found between the genogroup and presenting symptoms (OR 0.96; 95% CI 0.41-2.26). Viral loads were higher in symptomatic than in asymptomatic patients (p = 0.001)

First-line treatment in lymphomatoid papulosis: a retrospective multicentre study

Background: Data regarding response to treatment in lymphomatoid papulosis (LyP) are scarce. Aim: To assess the daily clinical practice approach to LyP and the response to first-line treatments. Methods: This was a retrospective study enrolling 252 patients with LyP. Results: Topical steroids, methotrexate and phototherapy were the most common first-line treatments, prescribed for 35%, 20% and 14% of the patients, respectively. Complete response (CR) was achieved in 48% of treated patients. Eczematous lesions significantly increased relative risk (RR) of not achieving CR (RR = 1.76; 95% CI 1.16-2.11). Overall median time to CR was 10 months (95% CI 6-13 months), and 78% of complete responders showed cutaneous relapse; both results were similar for all treatment groups (P > 0.05). Overall estimated median disease-free survival (DFS) was 11 months (95% CI 9-13 months) but DFS for patients treated with phototherapy was 23 months (95% CI 10-36 months; P < 0.03). Having the Type A LyP variant (RR = 2.04; 95% CI 0.96-4.30) and receiving a first-line treatment other than phototherapy (RR = 5.33; 95% CI 0.84-33.89) were significantly associated with cutaneous early relapse. Of the 252 patients, 31 (13%) had associated mycosis fungoides unrelated to therapeutic approach, type of LyP or T-cell receptor clonality. Conclusions: Current epidemiological, clinical and pathological data support previous results. Topical steroids, phototherapy and methotrexate are the most frequently prescribed first-line treatments. Although CR and cutaneous relapse rates do not differ between them, phototherapy achieves a longer DFS. Presence of Type A LyP and use of topical steroid or methotrexate were associated with an increased risk of early relapse

Detection of norovirus in saliva samples from acute gastroenteritis cases and asymptomatic subjects : Association with age and higher shedding in stool

Norovirus infections are a leading cause of acute gastroenteritis outbreaks worldwide and across all age groups, with two main genogroups (GI and GII) infecting humans. The aim of our study was to investigate the occurrence of norovirus in saliva samples from individuals involved in outbreaks of acute gastroenteritis in closed and semiclosed institutions, and its relationship with the virus strain, virus shedding in stool, the occurrence of symptoms, age, and the secretor status of the individual. Epidemiological and clinical information was gathered from norovirus outbreaks occurring in Catalonia, Spain during 2017-2018, and stool and saliva samples were collected from affected and exposed resident individuals and workers. A total of 347 saliva specimens from 25 outbreaks were analyzed. Further, 84% of individuals also provided a paired stool sample. For GII infections, norovirus was detected in 17.9% of saliva samples from symptomatic cases and 5.2% of asymptomatic individuals. Positivity in saliva occurred in both secretors and nonsecretors. None of the individuals infected by norovirus GI was positive for the virus in saliva. Saliva positivity did not correlate with any of the studied symptoms but did correlate with age ≥ 65 years old. Individuals who were positive in saliva showed higher levels of virus shedding in stool. Mean viral load in positive saliva was 3.16 ± 1.08 log10 genome copies/mL, and the predominance of encapsidated genomes was confirmed by propidium monoazide (PMA)xx-viability RTqPCR assay. The detection of norovirus in saliva raises the possibility of oral-to-oral norovirus transmission during the symptomatic phase and, although to a lesser extent, even in cases of asymptomatic infections

"Nested" cryptic diversity in a widespread marine ecosystem engineer: a challenge for detecting biological invasions

<p>Abstract</p> <p>Background</p> <p>Ecosystem engineers facilitate habitat formation and enhance biodiversity, but when they become invasive, they present a critical threat to native communities because they can drastically alter the receiving habitat. Management of such species thus needs to be a priority, but the poorly resolved taxonomy of many ecosystem engineers represents a major obstacle to correctly identifying them as being either native or introduced. We address this dilemma by studying the sea squirt <it>Pyura stolonifera</it>, an important ecosystem engineer that dominates coastal communities particularly in the southern hemisphere. Using DNA sequence data from four independently evolving loci, we aimed to determine levels of cryptic diversity, the invasive or native status of each regional population, and the most appropriate sampling design for identifying the geographic ranges of each evolutionary unit.</p> <p>Results</p> <p>Extensive sampling in Africa, Australasia and South America revealed the existence of "nested" levels of cryptic diversity, in which at least five distinct species can be further subdivided into smaller-scale genetic lineages. The ranges of several evolutionary units are limited by well-documented biogeographic disjunctions. Evidence for both cryptic native diversity and the existence of invasive populations allows us to considerably refine our view of the native versus introduced status of the evolutionary units within <it>Pyura stolonifera </it>in the different coastal communities they dominate.</p> <p>Conclusions</p> <p>This study illustrates the degree of taxonomic complexity that can exist within widespread species for which there is little taxonomic expertise, and it highlights the challenges involved in distinguishing between indigenous and introduced populations. The fact that multiple genetic lineages can be native to a single geographic region indicates that it is imperative to obtain samples from as many different habitat types and biotic zones as possible when attempting to identify the source region of a putative invader. "Nested" cryptic diversity, and the difficulties in correctly identifying invasive species that arise from it, represent a major challenge for managing biodiversity.</p

SARS-CoV-2 Catalonia contact tracing program : evaluation of key performance indicators

Background: Guidance on SARS-CoV-2 contact tracing indicators have been recently revised by international public health agencies. The aim of the study is to describe and analyse contact tracing indicators based on Catalonia's (Spain) real data and proposing to update them according to recommendations. Methods: Retrospective cohort analysis including Catalonia's contact tracing dataset from 20 May until 31 December 2020. Descriptive statistics are performed including sociodemographic stratification by age, and differences are assessed over the study period. Results: We analysed 923,072 contacts from 301,522 SARS-CoV-2 cases with identified contacts (67.1% contact tracing coverage). The average number of contacts per case was 4.6 (median 3, range 1-243). A total of 403,377 contacts accepted follow-up through three phone calls over a 14-day quarantine period (84.5% of contacts requiring follow-up). The percentage of new cases declared as contacts 14 days prior to diagnosis evolved from 33.9% in May to 57.9% in November. All indicators significantly improved towards the target over time (p < 0.05 for all four indicators). Conclusions: Catalonia's SARS-CoV-2 contact tracing indicators improved over time despite challenging context. The critical revision of the indicator's framework aims to provide essential information in control policies, new indicators proposed will improve system delay's follow-up. The study provides information on COVID-19 indicators framework experience from country's real data, allowing to improve monitoring tools in 2021-2022. With the SARS-CoV-2 pandemic being so harmful to health systems and globally, is important to analyse and share contact tracing data with the scientific community

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants