Impact of Retinopathy of Prematurity on Visual Motor Integration.

BACKGROUND: Preterm infants are at risk for neurodevelopmental deficits. An association between retinopathy of prematurity (ROP) and impaired cognitive outcome has already been described. However, less is known about the impact of ROP on visual motor integration (VMI), which is a prerequisite not only for fine motor abilities but also for further school skills. Therefore, the aim of this study was to retrospectively investigate the impact of ROP on VMI at preschool age. METHODS: The study was conducted at the Medical University of Vienna, including patients born between January 2009 and December 2014 with a gestational age of less than 30 weeks and/or a birth weight of less than 1,500 g. VMI was determined by Beery-Buktenica Developmental Test of Visual Motor Integration (Beery VMI) at the age of 5 years. RESULTS: Out of 1,365 patients, 353 met inclusion criteria for this study. Two hundred sixteen of them had no ROP, while 137 had ROP (stage 1: n = 23, stage 2: n = 74, stage 3: n = 40). Mean value of the Beery VMI score was significantly lower in the ROP group compared to the No-ROP group (90 ± 16 vs. 99 ± 14; p < 0.01). By correcting for other important medical conditions, ROP still had a significant impact on Beery VMI score (p < 0.01). Particularly, lower scores were found for stage 2 (p < 0.01) and stage 3 (p < 0.01). CONCLUSION: Beery VMI scores were significantly lower in preterm infants with ROP stage 2 and 3 than in infants without ROP. This study shows the negative impact of ROP on VMI skills at preschool age, even after adjustment for key demographic and medical characteristics.

Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17.

Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 Å resolution. DmxA structure contains several features distinct from known members of haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.

CAVER Analyst 2.0: analysis and visualization of channels and tunnels in protein structures and molecular dynamics trajectories.

Motivation: Studying the transport paths of ligands, solvents, or ions in transmembrane proteins and proteins with buried binding sites is fundamental to the understanding of their biological function. A detailed analysis of the structural features influencing the transport paths is also important for engineering proteins for biomedical and biotechnological applications. Results: CAVER Analyst 2.0 is a software tool for quantitative analysis and real-time visualization of tunnels and channels in static and dynamic structures. This version provides the users with many new functions, including advanced techniques for intuitive visual inspection of the spatiotemporal behavior of tunnels and channels. Novel integrated algorithms allow an efficient analysis and data reduction in large protein structures and molecular dynamic simulations. Availability and implementation: CAVER Analyst 2.0 is a multi-platform standalone Java-based application. Binaries and documentation are freely available at www.caver.cz. Supplementary information: Supplementary data are available at Bioinformatics online.

Wedelolactone Acts as Proteasome Inhibitor in Breast Cancer Cells.

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells.

Enzyme Tunnels and Gates As Relevant Targets in Drug Design.

Many enzymes contain tunnels and gates that are essential to their function. Gates reversibly switch between open and closed conformations and thereby control the traffic of small molecules-substrates, products, ions, and solvent molecules-into and out of the enzyme's structure via molecular tunnels. Many transient tunnels and gates undoubtedly remain to be identified, and their functional roles and utility as potential drug targets have received comparatively little attention. Here, we describe a set of general concepts relating to the structural properties, function, and classification of these interesting structural features. In addition, we highlight the potential of enzyme tunnels and gates as targets for the binding of small molecules. The different types of binding that are possible and the potential pharmacological benefits of such targeting are discussed. Twelve examples of ligands bound to the tunnels and/or gates of clinically relevant enzymes are used to illustrate the different binding modes and to explain some new strategies for drug design. Such strategies could potentially help to overcome some of the problems facing medicinal chemists and lead to the discovery of more effective drugs.

Discovery of Novel Haloalkane Dehalogenase Inhibitors.

Haloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 µM and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization.

Wedelolactone induces growth of breast cancer cells by stimulation of estrogen receptor signalling.

Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors, 5-lipoxygenase and topoisomerase IIα. It is cytotoxic to breast, prostate, pituitary and myeloma cancer cell lines in vitro at µM concentrations. In this study, however, a novel biological activity of nM dose of wedelolactone was demonstrated. Wedelolactone acts as agonist of estrogen receptors (ER) α and ß as demonstrated by transactivation of estrogen response element (ERE) in cells transiently expressing either ERα or ERß and by molecular docking of this coumestan into ligand binding pocket of both ERα and ERß. In breast cancer cells, wedelolactone stimulates growth of estrogen receptor-positive cells, expression of estrogen-responsive genes and activates rapid non-genomic estrogen signalling. All these effects can be inhibited by pretreatment with pure ER antagonist ICI 182,780 and they are not observed in ER-negative breast cancer cells. We conclude that wedelolactone acts as phytoestrogen in breast cancer cells by stimulating ER genomic and non-genomic signalling pathways.

Mechanism-based discovery of novel substrates of haloalkane dehalogenases using in silico screening.

Substrate specificity is a key feature of enzymes determining their applicability in biomaterials and biotechnologies. Experimental testing of activities with novel substrates is a time-consuming and inefficient process, typically resulting in many failures. Here, we present an experimentally validated in silico method for the discovery of novel substrates of enzymes with a known reaction mechanism. The method was developed for a model system of biotechnologically relevant enzymes, haloalkane dehalogenases. On the basis of the parametrization of six different haloalkane dehalogenases with 30 halogenated substrates, mechanism-based geometric criteria for reactivity approximation were defined. These criteria were subsequently applied to the previously experimentally uncharacterized haloalkane dehalogenase DmmA. The enzyme was computationally screened against 41,366 compounds, yielding 548 structurally unique compounds as potential substrates. Eight out of 16 experimentally tested top-ranking compounds were active with DmmA, indicating a 50% success rate for the prediction of substrates. The remaining eight compounds were able to bind to the active site and inhibit enzymatic activity. These results confirmed good applicability of the method for prioritizing active compounds-true substrates and binders-for experimental testing. All validated substrates were large compounds often containing polyaromatic moieties, which have never before been considered as potential substrates for this enzyme family. Whereas four of these novel substrates were specific to DmmA, two substrates showed activity with three other tested haloalkane dehalogenases, i.e., DhaA, DbjA, and LinB. Additional validation of the developed screening strategy with the data set of over 200 known substrates of Candida antarctica lipase B confirmed its applicability for the identification of novel substrates of other biotechnologically relevant enzymes with an available tertiary structure and known reaction mechanism.

Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites.

The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.

CAVER Analyst 1.0: graphic tool for interactive visualization and analysis of tunnels and channels in protein structures.

UNLABELLED: The transport of ligands, ions or solvent molecules into proteins with buried binding sites or through the membrane is enabled by protein tunnels and channels. CAVER Analyst is a software tool for calculation, analysis and real-time visualization of access tunnels and channels in static and dynamic protein structures. It provides an intuitive graphic user interface for setting up the calculation and interactive exploration of identified tunnels/channels and their characteristics. AVAILABILITY AND IMPLEMENTATION: CAVER Analyst is a multi-platform software written in JAVA. Binaries and documentation are freely available for non-commercial use at http://www.caver.cz.