CHK1 expression in gastric cancer is modulated by p53 and RB1/E2F1: Implications in chemo/radiotherapy response

Radiation has a limited but relevant role in the adjuvant therapy of gastric cancer (GC) patients. Since Chk1 plays a critical function in cellular response to genotoxic agents, we aimed to analyze the role of Chk1 in GC as a biomarker for radiotherapy resistance. We analyzed Chk1 expression in AGS and MKN45 human GC cell lines by RT-QPCR and WB and in a small cohort of human patient’s samples. We demonstrated that Chk1 overexpression specifically increases resistance to radiation in GC cells. Accordingly, abrogation of Chk1 activity with UCN-01 and its expression with shChk1 increased sensitivity to bleomycin and radiation. Furthermore, when we assessed Chk1 expression in human samples, we found a correlation between nuclear Chk1 accumulation and a decrease in progression free survival. Moreover, using a luciferase assay we found that Chk1’s expression is controlled by p53 and RB/E2F1 at the transcriptional level. Additionally, we present preliminary data suggesting a posttranscriptional regulation mechanism, involving miR-195 and miR-503, which are inversely correlated with expression of Chk1 in radioresistant cells. In conclusion, Chk1/microRNA axis is involved in resistance to radiation in GC, and suggests Chk1 as a potential tool for optimal stratification of patients susceptible to receive adjuvant radiotherapy after surgeryThis work was supported by Instituto de Salud Carlos III–Fondo de Investigación Sanitaria (PS09/1988 to ISP; PI11-00949, pI014-1495 and Feder Funds to RP); Comunidad Autónoma de Madrid-Universidad Autónoma de Madrid (CCG10-UAM/BIO-5871 to ISP); Fundación Leticia Castillejo Castillo and Ministerio de Ciencia e Innovación (SAF2012-30862 to RSP), Spain

Surface Analysis Insight Note: Observations relating to photoemission peak shapes, oxidation state, and chemistry of titanium oxide films

It is common practice to describe the coordination of metal atoms in a binding configuration with their nearest neighbors in terms of oxidation state, a measure by which the number of electrons redistributed between atoms forming chemical bonds. In XPS terms, change to an oxidation state is commonly inferred by correlating photoemission signal with binding energy. The assumption, when classifying photoemission signals into distinct spectral shapes, is that a distribution of intensities shifted to lower binding energy is evidence of a reduction in oxidation state. In this Insight note, we raise the prospect that changes in photoemission peak shape may occur without obvious changes, determined by XPS in stoichiometry for a material. It is well known that TiO2 measured by XPS yields reproducible Ti 2p photoemission peaks. However, on exposing TiO2 to ion beams, Ti 2p photoemission evolves to complex distributions in intensity, which are particularly difficult to analyze by traditional fitting of bell‐shaped curves to these data. For these reasons, in this Insight note, a thin film of TiO2 deposited on a silicon substrate is chosen for analysis by XPS and linear algebraic techniques. Alterations in spectral shapes created from modified TiO2, which might be interpreted as the change in oxidation state, are assessed in terms of relative proportions of titanium to oxygen. It is found through detailed analysis of spectra that quantification by XPS, using procedures routinely used in practice, is not in accord with the typical interpretations of photoemission shapes. The data processing methods used and results presented in this work are of particular relevance to elucidating fundamental phenomena governing the surface evolution of materials‐enabled energy processes where cyclic/non‐steady usage changes the nature of bonding, especially in the presence of contaminants

Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis

Enzyme engineering has allowed not only the de novo creation of active sites catalysing known biological reactions with rates close to diffusion limits, but also the generation of abiological sites performing new-to-nature reactions. However, the catalytic advantages of engineering multiple active sites into a single protein scaffold are yet to be established. Here, we report on proteins with two active sites of biological and/or abiological origin, for improved natural and non-natural catalysis. The approach increased the catalytic properties, such as enzyme efficiency, substrate scope, stereoselectivity and optimal temperature window, of an esterase containing two biological sites. Then, one of the active sites was metamorphosed into a metal-complex chemocatalytic site for oxidation and Friedel–Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a single protein. The transformations of 1-naphthyl acetate into 1,4-naphthoquinone (conversion approx. 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (≥83%; e.e. >99.9%) were achieved in one pot with this artificial multifunctional metalloenzyme.This work was funded by grant ‘INMARE’ from the European Union’s Horizon 2020 (grant agreement no. 634486), grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET), CTQ2016-79138-R, BIO2016-76601-C3-1-R, BIO2016-76601-C3-3-R, BIO2017-85522-R, RTI2018-095166-B-I00 and RTI2018-095090-B-100 from the Ministerio de Economía y Competitividad, the Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI), the Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (EU). P.N.G. and R.B. acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC; grant No. BB/M029085/1) and the Centre of Environmental Biotechnology Project and the Supercomputing Wales project, which are partly funded by the European Regional Development Fund (ERDF) through the Welsh Government. The authors gratefully acknowledge the financial support provided by the ERDF. C.C. thanks the Ministerio de Economía y Competitividad and FEDER for a Ph.D. fellowship (Grant BES-2015-073829). J.L.G.-A. thanks the support of the Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044). I.C.-R. thanks the Regional Government of Madrid for a fellowship (PEJ_BIO_AI_1201). The authors would like to acknowledge S. Ciordia and M. C. Mena for MALDI-TOF/TOF analysis. We thank the staff of both the European Synchrotron Radiation Facility (ESRF, Grenoble, France), for providing access and technical assistance at beamline ID30A-1/MASSIf-1, and the Synchrotron Radiation Source at Alba (Barcelona, Spain), for assistance at BL13-XALOC beamline. The authors would also like to acknowledge M. J. Vicente and M. A. Pascual at the Servicio Interdepartamental de Investigación (SIDI) of the Autonomous University of Madrid for the ESI-MS analyses

Decoding the ocean's microbiological secrets for marine enzyme biodiscovery

A global census of marine microbial life has been underway over the past several decades. During this period, there have been scientific breakthroughs in estimating microbial diversity and understanding microbial functioning and ecology. It is estimated that the ocean, covering 71% of the earth's surface with its estimated volume of about 2 x 10(18) m(3) and an average depth of 3800 m, hosts the largest population of microbes on Earth. More than 2 million eukaryotic and prokaryotic species are thought to thrive both in the ocean and on its surface. Prokaryotic cell abundances can reach densities of up to 10(12) cells per millilitre, exceeding eukaryotic densities of around 10(6) cells per millilitre of seawater. Besides their large numbers and abundance, marine microbial assemblages and their organic catalysts (enzymes) have a largely underestimated value for their use in the development of industrial products and processes. In this perspective article, we identified critical gaps in knowledge and technology to fast-track this development. We provided a general overview of the presumptive microbial assemblages in oceans, and an estimation of what is known and the enzymes that have been currently retrieved. We also discussed recent advances made in this area by the collaborative European Horizon 2020 project 'INMARE'

Surface science insight note: A linear algebraic approach to elucidate native films on Fe3O4 surface

Standard materials are often used to obtain spectra that can be compared to those from unknown samples. Spectra measured from these known substances are also used as a means of computing sensitivity factors to allow quantification by X‐ray photoelectron spectroscopy (XPS) of less well‐defined materials. Spectra from known materials also provide line shapes suitable for inclusion in spectral models which, when fitted to spectra, permit the chemical state for a sample to be assessed. Both types of information depend on isolating photoemission signals from the inelastically scattered signal. In this Insight note, technical issues associated with the use of XPS of as received Fe3O4 powder sample surface are discussed. The Insight note is designed to show how linear algebraic techniques applied to data collected from a sample marketed as pure Fe3O4 powder are used to verify that XPS has been performed on chemistry representative of the sample. The methods described in this Insight note can further be utilized in elucidating complex XPS data obtained from thin films formed or evolved during cyclic/non‐steady use of complex (electro)catalyst surfaces, especially in the presence of contaminants

Surface science insight note: Imaging X‐ray photoelectron spectroscopy

Quantification of X‐ray photoelectron spectroscopy (XPS) data is often limited by the heterogeneous nature of the material surface. However, it is often the case that heterogeneous material contains areas within the analyzed area that are effectively homogeneous. In this Insight note, concepts, and methods used to analyze both XPS data are presented to extract both spatial and spectral information from heterogeneous surfaces. These concepts and methods are applied to a specific material surface that contains three chemical compounds separated spatially. The analysis entails converting XPS image data to spectral data and is designed to highlight the potential of XPS imaging in revealing compositional information correlation with spatial information. Properties of algorithms used to evaluate XPS images and spectra are described to outline their application to image data. A case study of an imaging XPS data set is presented that demonstrates how poor signal‐to‐noise images, where the signal is recorded for 4 s per image, are still open to analysis yielding useful information. Ultimately, the methods presented here will aid in interpreting complex XPS data obtained from spatially complex materials often obtained during extensive cycling, such as conventional or electrocatalysts

Combining PCA and nonlinear fitting of peak models to re-evaluate C 1s XPS spectrum of cellulose

creation of new chemistry not present in the as-received sample. While improvements in instrumentation may be seen in general as beneficial to surface science, recent studies have shown that the consequences for some materials are detrimental. In this work, these problems are illustrated through an analysis of cellulose spectra obtained during a degradation study. C 1s spectra are decomposed into two well-formed component curves that are open to chemical interpretation. In particular, a component-curve representative of pure cellulose is obtained as well as a second component curve that implies cellulose is degraded through the creation of carbon chemistry involving Csingle bondO, Cdouble bondO and Osingle bondCdouble bondO. Since cellulose is a crystalline material, formed through the alignment of molecules under the influence of hydrogen bonds, the analysis and findings presented in this paper are relevant to any material analyzed by XPS whose properties are dependent on hydrogen bonds. The analysis techniques are based on an informed vectorial approach, which extracts directly from data spectral shapes that are used to monitor sample degradation via linear least squares optimization. Related mathematics of Principal Component Analysis and linear analysis are presented

Surface chemistry of ion beam modified native titania/Ti interfaces examined using X-ray photoelectron spectroscopy

It is often assumed in X-ray Photoelectron Spectroscopy that binding energy shifts are synonymous with changes in the chemical state of an atom and the chemical state can be described in terms of oxidation state and stoichiometry of the elements in a material that correlates with photoemission peaks. However, when an atom is bonded into a crystal lattice, the shapes and binding energy of photoemission may not match the expected stoichiometry when measured by XPS, even though shifts in binding energy suggest new oxidation states. In this work, a set of experiments is presented, in which Ar+ and He+ ions of different energies modify the native oxide on a titanium foil which yields XPS spectra that are not easily open to analysis by conventional peak models. In the course of analyzing these complex photoemission data by linear algebraic methods, the prospect emerged that XPS is suggesting that sputtering a native oxide may be providing insight into structural perturbation in addition to the stoichiometry-type changes to the native oxide