Archaeology of the sky. Astronomical orientations in Protohistoric buildings of the south of the Iberian Peninsula

Presentamos un análisis arqueoastronómico de la orientación de seis santuarios y edificios de carácter público protohistóricos en sitios arqueológicos situados en el mediodía de la Península Ibérica (Coria del Río, El Carambolo, Saltillo, Mesa de Setefilla, Tejada la Vieja y El Oral). El estudio, el primero de este tipo que se realiza sobre una muestra de santuarios del Hierro Antiguo en esta área geográfica, se basa en la medida precisa de las orientaciones definidas por los edificios y el análisis del horizonte que les rodea y revela claros vínculos astronómicos. En particular, los edificios de culto muestran unas características similares y una orientación hacia un acimut de 55°. Las regularidades encontradas parecen sugerir la posible existencia de credos y rituales religiosos relacionados con posiciones singulares de los astros principales visibles a simple vista, como el Sol, la Luna o Venus.We present an archaeoastronomical analysis of the orientations of six protohistoric sanctuaries and public buildings located in archaeological sites of the south of the Iberian Peninsula (Coria del Río, El Carambolo, Saltillo, Mesa de Setefilla, Tejada la Vieja and El Oral). This study, the first of its kind conducted on a sample of Early Iron Age sanctuaries in this geographic area, is based on the accurate measurement of the orientations defined by the buildings and the analysis of the horizon around them and reveals clear astronomical relations. In particular, religious buildings show similar characteristics and orientation towards an azimuth of 55°. The regularities found seem to suggest the existence of religious beliefs and rituals linked to singular positions of the brightest celestial bodies as the Sun, Moon or Venus

Biología estructural de la maquinaria de remodelado y degradación de la pared bacteriana: mecanismos de activación de la amidasa AmpD y el enzibiótico GP61

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-11-201

Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg-1) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg-1), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.Financial and logistic support from Colombian Universidad del Valle and COLCIENCIAS (CI 71083-Grant 745-2016-Project 110671250425), Spanish CICYT project BIO-2005-6018576, BFU2017-90030-P, and BFU2011-25326, B. Di G. In addition, thanks to the Spanish MINECO for a FPU fellowship.S

Reaction products and the X-ray structure of AmpDh2, a virulence determinant of Pseudomonas aeruginosa

4 pags, 4 figs. -- Supporting Information is available at the Publisher web.The zinc protease AmpDh2 is a virulence determinant of Pseudomonas aeruginosa, a problematic human pathogen. The mechanism of how the protease manifests virulence is not known, but it is known that it turns over the bacterial cell wall. The reaction of AmpDh2 with the cell wall was investigated, and nine distinct turnover products were characterized by LC/MS/MS. The enzyme turns over both the cross-linked and noncross-linked cell wall. Three high-resolution X-ray structures, the apo enzyme and two complexes with turnover products, were solved. The X-ray structures show how the dimeric protein interacts with the inner leaflet of the bacterial outer membrane and that the two monomers provide a more expansive surface for recognition of the cell wall. This binding surface can accommodate the 3D solution structure of the cross-linked cell wall. © 2013 American Chemical Society.This work was supported by a grant from the NIH (GM61629) and by grants BFU2011-25326 (the Spanish Ministry of Economy and Competitiveness) and S2010/BMD-2457 (the Government of Community of Madrid). The Mass Spectrometry & Proteomics Facility of the University of Notre Dame is supported by grant CHE0741793 from the NSF

Structural basis for the broad specificity of a new family of amino-acid racemases

Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical d-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family membersResearch in the Cava laboratory is supported by the MINECO, Spain (RYC-2010-06241), Universidad Autonoma de Madrid (UAM-38) and by the Knut and Alice Wallenberg Foundation (KAW). Additionally, this work was supported by the BFU2011-25326 MEC grant (JAH), by the S2010/BMD-2457 grant from CAM (JAH) and by HHMI (MKW

Crystal structures of bacterial peptidoglycan amidase AmpD and an unprecedented activation mechanism

9 pags, 5 figs, 2 tabsAmpD is a cytoplasmic peptidoglycan (PG) amidase involved in bacterial cell-wall recycling and in induction of β-lactamase, a key enzyme of β-lactam antibiotic resistance. AmpD belongs to the amidase-2 family that includes zinc-dependent amidases and the peptidoglycan-recognition proteins (PGRPs), highly conserved pattern-recognition molecules of the immune system. Crystal structures of Citrobacter freundii AmpD were solved in this study for the apoenzyme, for the holoenzyme at two different pH values, and for the complex with the reaction products, providing insights into the PG recognition and the catalytic process. These structures are significantly different compared with the previously reported NMR structure for the same protein. TheNMRstructure does not possess an accessible active site and shows the protein in what is proposed herein as an inactive "closed" conformation. The transition of the protein from this inactive conformation to the active "open" conformation, as seen in the x-ray structures, was studied by targeted molecular dynamics simulations, which revealed large conformational rearrangements (as much as 17 Å ) in four specific regions representing one-third of the entire protein. It is proposed that the large conformational change that would take the inactive NMR structure to the active x-ray structure represents an unprecedented mechanism for activation of AmpD. Analysis is presented to argue that this activation mechanism might be representative of a regulatory process for other intracellular members of the bacterial amidase-2 family of enzymes. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported, in whole or in part, by the National Institutes of Health. This work was also supported by grants from the Spanish Ministry of Science and Technology (BFU2008-01711), EU-CP223111 (CARE-PNEUMO, European Union), and the COMBACT program (S-BIO-0260/2006). We acknowledge the Spanish Ministerio de Ciencia e Innovación (PI201060E013) and Consejo Superior de Investigaciones Científicas for financial support and for provision of synchrotron radiation facilitie

Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins

15 p.-8 fig.-2 tab.PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.This research was funded by the following sources: Grants PID2019-105126RB-I00, PID2022-139209OB-C21 (MCIN/AEI/10.13039/501100011033/and ERDF A way of making Europe), TED2021-129747B-C22 (AEI/10.13039/501100011033/NextGenerationEU/PRTR) and CIBER-Consorcio Centro de Investigación Biomédica en Red (CIBERES, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain) to JMS; grants PID2020-115331GB-100 funded by MCIN/AEI/10.13039/501100011033 and CRSII5_198737/1 (Swiss National Science Foundation) to JAH; grant PID2021-128751NB-I00 (MICINN/AEI/FEDER/UE) to IU, and grant RYC2021-030916-I by the Spanish Agencia Estatal de Investigación to RM. VMH-R was supported by a FPU PhD fellowship from Spanish Ministerio de Educación y Ciencia.Peer reviewe

Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation

16 pags, 7 figs . -- The online version contains supplementary movie1: https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-021-27657-y/MediaObjects/41467_2021_27657_MOESM3_ESM.mp4Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.This research was supported by the grant PID2019-105872GB-I00/AEI/10.13039/ 501100011033 from the Spanish Ministry of Science and Innovation to J.M.V. and J.C. as well as FRIMEDBIO (261826) from the Research Council of Norway to A.M.; the Western Norway Regional Health Authorities (912246 to A.M. and 912264 to R.K.), the K.G.Peer reviewe

Health-related quality of life with palbociclib plus endocrine therapy versus capecitabine in postmenopausal patients with hormone receptor–positive metastatic breast cancer: Patient-reported outcomes in the PEARL study

Background: The PEARL study showed that palbociclib plus endocrine therapy (palbociclib/ET) was not superior to capecitabine in improving progression-free survival in postmenopausal patients with metastatic breast cancer resistant to aromatase inhibitors, but was better tolerated. This analysis compared patient-reported outcomes. Patients and methods: The PEARL quality of life (QoL) population comprised 537 patients, 268 randomised to palbociclib/ET (exemestane or fulvestrant) and 269 to capecitabine. Patients completed the European Organisation for Research and Treatment of Cancer QLQC30 and QLQ-BR23 and EQ-5D-3L questionnaires. Changes from the baseline and time to deterioration (TTD) were analysed using linear mixed-effect and stratified Cox regression models, respectively. Results: Questionnaire completion rate was high and similar between treatment arms. Significant differences were observed in the mean change in global health status (GHS)/QoL scores from the baseline to cycle 3 (2.9 for palbociclib/ET vs.-2.1 for capecitabine (95% confidence interval [CI], 1.4-8.6; P = 0.007). The median TTD in GHS/QoL was 8.3 months for palbociclib/ET versus 5.3 months for capecitabine (adjusted hazard ratio, 0.70; 95% CI, 0.55-0.89; P = 0.003). Similar improvements for palbociclib/ET were also seen for other scales as physical, role, cognitive, social functioning, fatigue, nausea/vomiting and appetite loss. No differences were observed between the treatment arms in change from the baseline in any item of the EQ-5D-L3 questionnaire as per the overall index score and visual analogue scale. Conclusion: Patients receiving palbociclib/ET experienced a significant delay in deterioration of GHS/QoL and several functional and symptom scales compared with capecitabine, providing additional evidence that palbociclib/ET is better tolerated. Trial registration number: NCT02028507 (ClinTrials.gov). EudraCT study number: 2013-003170-27. 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

PAM50 proliferation score as a predictor of weekly paclitaxel benefit in breast cancer

To identify a group of patients who might benefit from the addition of weekly paclitaxel to conventional anthracycline-containing chemotherapy as adjuvant therapy of node-positive operable breast cancer. The predictive value of PAM50 subtypes and the 11-gene proliferation score contained within the PAM50 assay were evaluated in 820 patients from the GEICAM/9906 randomized phase III trial comparing adjuvant FEC to FEC followed by weekly paclitaxel (FEC-P). Multivariable Cox regression analyses of the secondary endpoint of overall survival (OS) were performed to determine the significance of the interaction between treatment and the (1) PAM50 subtypes, (2) PAM50 proliferation score, and (3) clinical and pathological variables. Similar OS analyses were performed in 222 patients treated with weekly paclitaxel versus paclitaxel every 3 weeks in the CALGB/9342 and 9840 metastatic clinical trials. In GEICAM/9906, with a median follow up of 8.7 years, OS of the FEC-P arm was significantly superior compared to the FEC arm (unadjusted HR = 0.693, p = 0.013). A benefit from paclitaxel was only observed in the group of patients with a low PAM50 proliferation score (unadjusted HR = 0.23, p < 0.001; and interaction test, p = 0.006). No significant interactions between treatment and the PAM50 subtypes or the various clinical–pathological variables, including Ki-67 and histologic grade, were identified. Finally, similar OS results were obtained in the CALGB data set, although the interaction test did not reach statistical significance (p = 0.109). The PAM50 proliferation score identifies a subset of patients with a low proliferation status that may derive a larger benefit from weekly paclitaxel. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10549-013-2416-2) contains supplementary material, which is available to authorized users