Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder characterised by symptoms of inattention, impulsivity and hyperactivity. The spontaneously hypertensive rat (SHR) is a well-characterised model of this disorder and has been shown to exhibit dopamine dysregulation, one of the hypothesised causes of ADHD. Since stress experienced in the early stages of life can have long-lasting effects on behaviour, it was considered that early life stress may alter development of the dopaminergic system and thereby contribute to the behavioural characteristics of SHR. It was hypothesized that maternal separation would alter dopamine regulation by the transporter (DAT) in ways that distinguish SHR from control rat strains.in vivo chronoamperometry to determine whether changes had occurred in striatal clearance of dopamine by DAT. The rate of disappearance of ejected dopamine was used as a measure of DAT function.SHR and control Wistar-Kyoto (WKY) rats were subjected to maternal separation for 3 hours per day from postnatal day 2 to 14. Rats were tested for separation-induced anxiety-like behaviour followed by -1) in SHR striatum. Consistent with this observation, the dopamine clearance time (T100) was increased in SHR. These results suggest that the chronic mild stress of maternal separation impaired the function of striatal DAT in SHR.Consistent with a model for ADHD, SHR were more active than WKY in the open field. SHR entered the inner zone more frequently and covered a significantly greater distance than WKY. Maternal separation increased the time that WKY spent in the closed arms and latency to enter the open arms of the elevated plus maze, consistent with other rat strains. Of note is that, maternal separation failed to produce anxiety-like behaviour in SHR. Analysis of the chronoamperometric data revealed that there was no difference in DAT function in the striatum of non-separated SHR and WKY. Maternal separation decreased the rate of dopamine clearance (kThe present findings suggest that maternal separation failed to alter the behaviour of SHR in the open field and elevated plus maze. However, maternal separation altered the dopaminergic system by decreasing surface expression of DAT and/or the affinity of DAT for dopamine, increasing the time to clear dopamine from the extracellular fluid in the striatum of SHR. Attention-deficit hyperactivity disorder (ADHD) is a developmental disorder characterised by three predominant symptoms of hyperactivity, impulsivity and inattention that can manifest in behavioural problems later in life ,2. ThougGenetic studies have focused chiefly on proteins involved in dopamine transmission , particuThe spontaneously hypertensive rat (SHR) is a well characterised genetic model of ADHD -11. ConsSimilar to patients with ADHD who displayed a variable number of tandem repeats in the 3'-untranslated region of the DAT gene ,18-21, SSince ADHD is considered to result from a complex interaction between small gene effects and the environment  rats werMaternal separation has been shown to have opposite effects on behaviour of SHR and WKY in the elevated plus maze . Materna® were purchased from Sigma Chemical Company . All other chemicals were of the highest standard and were purchased from Merck Chemicals (Germany). Carbon fibre electrodes were purchased from Quanteon LLC . Teflon-coated silver wire was purchased from A-M Systems .Ascorbic acid, dopamine-HCl, urethane and Nafionad libitum access to food and water. This study was conducted in accordance with international guidelines  and approved by the University of Cape Town Faculty of Health Sciences Animal Ethics Committee.Three month old male and female inbred WKY  and SHR rats  were obtained from the University of Cape Town Animal Unit and paired in cages for breeding. The date of birth of the litter was designated postnatal day 0 (P0). On P2, litters were culled to 8 pups to ensure equal nourishment during the early postnatal period. Male rats were preferentially selected to avoid the confounding effects of fluctuating hormones during the female oestrus cycle on brain function and behaviour in later experiments. This selection produced an average of 4.3 males and 3.7 females in WKY NMS litters, 4.6 males and 3 females in WKY MS litters, 6.3 males and 1.2 females in SHR NMS litters, and 5.2 males and 1.7 females in SHR MS litters. On P2, litters were designated as either maternally separated experimental rats or non-maternally separated control rats to give a total of 21 non-separated WKY; 22 maternally separated WKY; 20 non-separated SHR and 29 separated SHR. Rats were housed in the University of Cape Town satellite animal facility in cages with woodchip bedding and a 12 hour light/dark cycle (lights on at 06h00). Rats had The maternal separation protocol involved removal of the dam from the pups for 3 hours per day between 09h00 and 13h00 from P2 to P14 . Pups weOn P28, rats were taken to the behavioural facility and allowed to acclimatise to the behavioural testing room for a minimum of 1 hour prior to testing in the open field and elevated plus maze.The open field test measures total distance covered in a 100 cm × 100 cm (floor) × 50 cm  black box, as well as time spent and number of entries into the inner zone (70 cm × 70 cm) of the box. The tests were conducted in an isolated noise-free room. The lighting in the room was 50 lux. More time spent in the inner zone indicated increased exploration, and was defined as reduced anxiety ,42. The After the open field test, the rat was returned to the home cage and a minimum of 1 hour was allowed to pass before the rat was tested in the elevated the plus maze which has open and closed arms, elevated 50 cm above the ground. The animal was placed in the centre of the maze, facing an open arm and allowed to explore the maze for 5 minutes. Time spent in the open arms suggested decreased anxiety-like behaviour while time spent in the closed arms suggested increased anxiety-like behaviour . Gloves ® coating  was used to enhance the sensitivity and selectivity of the electrodes for dopamine over other electroactive molecules [2PO4, 40 mM Na2HPO4, 100 mM NaCl) to which a known amount of ascorbic acid and incremental amounts of dopamine were added to determine selectivity of the electrodes for dopamine and to generate a calibration curve of current versus dopamine concentration.High-speed in vivo chronoamperometric measurement of extracellular dopamine was carried out using the FAST-16 system  with Nafion-coated carbon fibre microelectrodes  ,45. Thisolecules . Prior tA square wave potential (0 to +0.55 V vs. a glass RE-5 Ag/AgCl reference electrode) was applied to the carbon fibre microelectrode for 100 ms to create a 5 Hz waveform repeated at 1 Hz intervals to cause the oxidation and subsequent reduction of dopamine at the microelectrode surface. Dopamine has a specific red/ox ratio (~0.7-0.9) which was used as a 'chemical fingerprint' to confirm specific dopamine recording . ChangesBetween P49 and P54 rats were deeply anesthetized with 25% urethane at a dose of 1.25 g/kg to produce non-recoverable terminal anaesthesia. Urethane was chosen because it affected the activity of neurotransmitter systems less than other anaesthetic agents . High-sp-1), and clearance time (T100) were measured  = 50.1, p < 0.0001, Mann-Whitney U test, p < 0.0001, Figure The total number of entries into the inner zone of the open field over the 5 minute test period was significantly greater for SHR compared to WKY rats  = 64.5, p < 0.0001, Mann-Whitney U test, p < 0.0001, Figure SHR spent less time in the closed arms of the elevated plus maze than WKY  = 21.1, p < 0.001, Mann-Whitney U test, p < 0.0001, Figure SHR displayed a shorter latency to enter the open arms of the elevated plus maze than WKY  = 27.6, p < 0.0001, Mann-Whitney U test, p < 0.0001, Figure -1, was similar in SHR and WKY striatum  = 1.3, p = 0.262) (Table -1) was observed in maternally separated SHR compared to non-separated SHR (Mann-Whitney U test, p < 0.05, Figure The rate of dopamine clearance by DAT, kConsistent with the behavioural characteristics of ADHD and in agreement with previous studies, SHR displayed increased exploratory activity and decreased measures of anxiety in the open field and elevated plus maze compared to WKY rats ,39. In a-1 was decreased and it took longer to clear a known amount of dopamine in SHR striatum, suggesting that the mild chronic developmental stress of maternal separation had decreased DAT efficiency and/or reduced surface expression of DAT. Postnatal development of dopamine terminals in the striatum, and hence DAT surface expression, occurs rapidly between P7 and P35 with smaller subsequent increases in DAT to reach adult levels at P60 [Maternal separation decreased the rate of clearance of exogenously applied dopamine in SHR striatum. The ks at P60 . The tims at P60  and incrs at P60 .These findings suggest that SHR have impaired DAT function, which is not apparent under normal conditions but becomes apparent when the individual is subjected to early developmental stress in the form of maternal separation. Environmental factors can therefore influence development of the dopaminergic system giving rise to impaired DAT function and possibly lead to ADHD-like symptoms. In fact, patients with ADHD have been shown to have decreased striatal DAT availability compared to controls . FurtherSHR displayed increased behavioural activation relative to WKY, consistent with SHR being a widely accepted model of ADHD. WKY displayed increased anxiety-like behaviour, consistent with the anxiogenic effects of maternal separation previously observed in WKY and other rat strains. Of note is that SHR did not display effects of maternal separation in behavioural tests of anxiety suggesting that SHR were resilient to the anxiogenic effects of chronic mild developmental stress. In contrast, maternal separation decreased the rate of clearance of dopamine in SHR striatum. SHR appear to have impaired DAT function, which is not apparent under normal conditions but becomes apparent when the individual is subjected to early developmental stress in the form of maternal separation.The authors have no competing interests except for the fact that GAG owns Quanteon, LLC, makers of the FAST recording system that was used to carry out measures of dopamine uptake.JSW contributed to the design of the study, carried out experimental procedures, analysed data and drafted the manuscript. JHH assisted with the experimental procedures and execution of the study. GAG, LAK and VAR contributed to the development, design and execution of the study, and analysis of the data. All authors have contributed to the drafting of the manuscript and have read and approved the final manuscript.


Molecular Brain has been accepted by Thomson Reuters for tracking and is due to receive its first official Impact Factor in June 2013.We are pleased to announce that We would like to thank our distinguished international editorial board for their efforts on behalf of the journal, and our publishers, BioMed Central, for their in-house contribution to the speed and efficiency with which manuscripts are processed. Most important of all, of course, are our authors and reviewers, and to them we extend our particular thanks.Molecular Brain has published over 175 articles covering a wide range of topics in basic and clinical molecular neurosciences. A rapid review process ensures that papers are published in a timely manner. Our average time from submission to the first decision is 24 days, even under our stringent peer review process. As an open access journal, all papers are made immediately and freely available and articles are being accessed more than ever, with an average of 18,000 accesses per month so far in 2012, and a record of 21,000 accesses in May 2012. Links to publications are now listed on the Molecular Brain Twitter account [Since it was launched in 2008, as the official journal of the Association for the study of Neurons and Diseases (AND), Each year the Molecular Brain Award is made to the corresponding author whose paper has received the most citations. This year the award was presented to Dr. Michael Schlossmacher (University of Ottawa) at the annual meeting of AND in Montreal Figure.A series of reviews on Molecules of Memory  is curreMolecular Brain.With the help of its readers and authors we look forward to the continuing growth of


EHP received more than 1,500 submissions during 2013, and nearly 500 of those papers were sent out for peer review. Each paper is evaluated by at least 2 reviewers for scientific quality and content. EHP is very grateful to the nearly 1,000 reviewers who were part of the peer-review process. A list of those reviewers is available on the journal’s website. EHP would also like to recognize our top Reviewers of the Year. These individuals completed at least 5 reviews during the year and received high ratings from the Associate Editors for the timeliness and quality of their reviews. The EHP 2013 Reviewers of the Year are Daniel Axelrad, John Balmes, Aaron Barchowsky, Adrian Barnett, David Bellinger, Joe Braun, Gerard Hoek, Matthew Longnecker, and C. Arden Pope III. EHP is grateful to these and all other reviewers who assisted us during 2013.


The research of multicomponent drugs, such as in Chinese Medicine, on both mechanism dissection and drug discovery is challenging, especially the approaches to systematically evaluating the efficacy at a molecular level. Here, we presented a network pharmacology-based approach to evaluating the efficacy of multicomponent drugs by genome-wide transcriptional expression data and applied it to Shenmai injection (SHENMAI), a widely used Chinese Medicine composed of red ginseng (RG) and Radix Ophiopogonis (RO) in clinically treating myocardial ischemia (MI) diseases. The disease network, MI network in this case, was constructed by combining the protein-protein interactions (PPI) involved in the MI enriched pathways. The therapeutic efficacy of SHENMAI, RG, and RO was therefore evaluated by a network parameter, namely, network recovery index (NRI), which quantitatively evaluates the overall recovery rate in MI network. The NRI of SHENMAI, RG, and RO were 0.876, 0.494, and 0.269 respectively, which indicated SHENMAI exerts protective effects and the synergistic effect of RG and RO on treating myocardial ischemia disease. The successful application of SHENMAI implied that the proposed network pharmacology-based approach could help researchers to better evaluate a multicomponent drug on a systematic and molecular level. Chinese Medicine, featured as having “multiple ingredients and multiple targets,” has been widely used to treat complex diseases for decades . So far,It would be promising to reveal a drug's efficacy via high throughput technology , such asRecently, network based analyses such as network pharmacology emerged and have become a powerful tool to systematically reveal complex biological relationships , 17. UnlPanax ginseng C. A. Mey, steamed and dry) and Radix Ophiopogonis  Ker-Gawl, root), is a Chinese Medicine that is widely used in clinically treating ischemic heart disease [In this study, for the first time, network pharmacology is introduced and applied to the evaluation of the efficacy of Shenmai injection (SHENMAI) in treating myocardial ischemia (MI). SHENMAI, composed of red ginseng . The HPLC fingerprinting assay was performed to estimate the batch-to-batch consistency of SHENMAI [SHENMAI no. 1107282) extracts of RG and RO, and the vehicle in the form of an injection, were supplied by 2 extractMale Sprague-Dawley rats (180–220 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. A rat model of myocardial infarction was induced by permanent occlusion of the left anterior descending coronary artery. Briefly, after anesthetized using diethyl ether, the rats were fixed with the hair over the heart picked, then underwent a left thoracotomy and were ligated on the left anterior descending coronary artery. The control (sham) group was given the same surgery but without ligation. SHENMAI and the vehicle were given via intraperitoneal injection for 7 days in rats with myocardial ischemia. In detail, 10 mL/kg of SHENMAI and the corresponding dose for RG and RO were given to the rats, respectively. The dosage of SHENMAI and its components used was calculated in accordance with clinical consumption and was confirmed by our preliminary study. On the eighth day, the rats were anaesthetized by intraperitoneal injection of 0.2 mL/100 g anesthetic, which was prepared by mixing diazepam and ketamine in the ratio of equality, for measuring echocardiography using a VisualSonics Vevo 770TM in vivo microimaging system equipped with an RMV-707B cardiovascular scanhead . Then the border between the infarct and noninfarct left ventricle area in the rat heart was harvested to extract mRNA for genome-wide transcriptomic analysis.Total RNA was extracted using TRIZOL Reagent  following the manufacturer's instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 . Qualified total RNA was further purified by RNeasy mini kit  and RNase-Free DNase Set . Total RNA was amplified, labeled, and purified by using GeneChip 3'IVT Express Kit  following the manufacturer's instructions to obtain biotin-labeled cRNA. Array hybridization and wash were performed using GeneChip Hybridization, Wash and Stain Kit  in Hybridization Oven 645  and Fluidics Station 450  following the manufacturer's instructions. Slides were scanned by GeneChip Scanner 3000  and Command Console Software 3.1  with default settings. Raw data were stored on ArrayTrack , a java-t-test with a threshold of P value <0.01 and fold change >1.5 was applied to find out the significant genes according to the microarray expression profiles. In this study, the significant genes differentially expressed between Myocardial ischemia (MI) group and control group were used for further pathway enrichment analysis.MI network was constructed based on the MI enriched pathways and PPI knowledge. To define MI enriched pathways, a widely used approach of Welch Pathway enrichment analysis was then applied to find the significant enriched pathways of these DEGs. ArrayTrack  was usedIn this study, the nodes of the MI network are the genes involved in the MI enriched pathways, and the edges are the protein-protein interactions between these genes. Since protein-protein interactions (PPIs) for rats were relatively few in number that could hardly reflect the overall network relationships between genes  was used as a quantitative index to evaluate the network perturbation recovery from MI to normal state by treatment. To calculate the NRI, the regulating score (RS) was first calculated to evaluate the influence of MI and SHENMAI on each node in the network, which was calculated by the average ratio of expression data between MI/treatment and control samples formula . In form. These 27 pathways involved 10 metabolism pathways, 10 cellular process pathways, 4 human disease pathways, 2 environmental information processing pathways, and DNA replication. For instance, The adipocytokine signaling pathway (KEGG id: rno04920) showed a P-value = 0.026, and dilated cardiomyopathy (KEGG id: rno05414) showed a P-value <1.0e − 8. A detailed list of the 27 significant pathways used in this study is in Supplement Table S1 .A combined algorithm with simple According to the KEGG database, a total of 1478 genes were involved in these 27 significant pathways. The Orthologene database in ArrayTrack was used to convert these genes from rat to human. As a result, 905 genes have been found to have at least one interaction with other genes (including self-loops), and all the 2618 interactions were involved. R2 = 0.868), indicating that MI the network was scale-free like The MI network was constructed based on these genes and interactions, and was visualized by Cytoscape (version 2.8.0)  Figure . In thisThe number of up-and downregulating nodes in the giant component by MI was 177 and 106; the number of up-and downregulating nodes by SHENMAI was 112 and 60, respectively. The result showed that the number of regulated nodes by SHENMAI was smaller than in MI. Regulating score maps of SHENMAI and MI were drawn based on the regulating score of the MI group and SHENMAI treatment , middle.up, Rrdown, and Rrall, were 0.9096, 0.9057, and 0.7787, respectively. So, the NRI of SHENMAI was 0.865. The NRI was used to quantitatively evaluate the efficacy of the drug, based on the ability of the drug to recover the network from MI to normal. The NRI was a value between 0 and 1, and a higher NRI represented a good efficacy in treating MI in our study. As mentioned above, the ratio of recovery regulation of SHENMAI, RrP = 0.0002 and 0.0006).We also calculated the NRI of RG, and RO, which were solely used to treat MI. The NRI of SHENMAI, RG and RO were 0.865, 0.425, and 0.271, respectively . Both RGP < 0.05) related to MI controls. However, individual treatment of RG and RO did not show significance to impair the cardiac function. In our study, LV function was used as the golden indicator to evaluate the drug efficacy of SHENMAI, RG, and RO in the development of cardiac failure after myocardial ischemia by using echocardiography after 7 days of treatment. As shown in The overall trends in EF and FS indicators of SHENMAI, RG, and RO were consistent with the NRI in the network based analysis, which showed SHENMAI had the best and significant efficacy performance according to both the echocardiography study and network based analysis, while RG and RO showed insignificant efficacy according to the echocardiography result. The accordance of the echocardiography result and NRI indicated that network pharmacology based analysis could reveal drug effects on treating MI on a molecula level related to phenotype indicators.In this study, we presented a network-based approach to evaluating the efficacy of Chinese Medicine using genome-wide transcriptional expression data. By constructing a specified biological network of MI and analyzing the network regulation, we quantitatively evaluated the efficacy of the multicomponent drug SHENMAI in treating MI. The result indicates that SHENMAI has a significant efficacy in treating MI, which was represented by its ability to repair the MI network. Furthermore, comparative analysis of SHENMAI and its components RG and RO indicated that RG and RO have the synergistic effect on treating MI. This result was further validated by LV function via echocardiography experiments. Based on our study, we believe that evaluating the efficacy of Chinese Medicine on a systematic and molecular level will be a trend in future drug research.Supplement Table: Detailed information of 27 enriched pathways. KEGG pathway analysis tools embedded in ArrayTrack were used for pathway analysis, and pathway with Fisher p-value <0.05 was identified as the enriched pathway. 27 enriched pathways were eventually identified and their detailed information was listed in Supplement Table .Click here for additional data file.


In the molecular sciences, only a small number of experts with specialized know-how are able to design interactive visualization applications, typically static computer programs that cannot easily be modified. Are there lessons to be learned from video games? Could their technology help us explore new molecular graphics ideas and render graphics developments accessible to non-specialists? This approach points to an extension of open computer programs, not only providing access to the source code, but also delivering an easily modifiable and extensible scientific research tool. In this work, we will explore these questions using the Unity3D game engine to develop and prototype a biological network and molecular visualization application for subsequent use in research or education. We have compared several routines to represent spheres and links between them, using either built-in Unity3D features or our own implementation. These developments resulted in a stand-alone viewer capable of displaying molecular structures, surfaces, animated electrostatic field lines and biological networks with powerful, artistic and illustrative rendering methods. We consider this work as a proof of principle demonstrating that the functionalities of classical viewers and more advanced novel features could be implemented in substantially less time and with less development effort. Our prototype is easily modifiable and extensible and may serve others as starting point and platform for their developments. A webserver example, standalone versions for MacOS X, Linux and Windows, source code, screen shots, videos and documentation are available at the address:  Scientific projects associated to these programs include ePMV Biology currently undergoes a rapid expansion, calling for tools to visualize huge and complex systems, such as macromolecular structures, -omics networks or even organs and organisms http://unity3d.com/), Unreal Engine , CryEngine (http://mycryengine.com/), Blender Game Engine . These engines are free for non-commercial use and very appealing for developing scientific applications.For many years, game developers have gathered routines and frameworks that can be reused for a wide range of games. These code blocks have been aggregated to provide toolkits, called game engines, embedding all the components required to create a game in one package Here, we focus on molecular and network visualization using surfaces, spheres and links between them. With this test case we assess whether by using a game engine the functionality of classical viewers could be reproduced and extended in substantially less time and with less development effort than with common tools. Current needs include the ability to display hundreds of thousands of distinguishable elements in interactive time, using classical representation schemes. To provide a realistic scenario, inspired by current research, we have implemented our own visualization algorithm based on latest hardware capabilities such as programmable Graphics Processing Units (GPUs) http://unity3d.com/) for its ability to deploy multi-platform applications with minimal programming effort. Furthermore, Unity3D provides an easy-to-use interface to develop 3D graphics applications using JavaScript, Boo – a python derived language - or C# code. Unity3D includes advanced functionalities to fully use the capacities of recent programmable graphics cards. This is possible employing optimized Cg functions We have chosen to test the Unity3D game engine  For structural data we use the Protein Data Bank We have tested graphical methods available in Unity3D as well as our HyperBalls representation based on ray casting -B,D).Unity3D provides an optimized set of graphical primitives for rendering. We use triangulated spheres, triangulated cubes and lines. Spheres and cubes display atoms (for molecular structures) or nodes (for networks); links between them are rendered using lines . In games, this visual method is commonly used to depict effects involving many small particles such as fire, smoke, clouds, snow, dust, etc. … For UnityMol, we have adapted Unity3D’s built-in particle system routine to paste spheres representing atoms on each square  always oriented perpendicular to the screen plane can be used to represent a single atom. An image of the atom sphere called sprite is pasted onto the square. This point-sprite method is very efficient to display many similar images and can be extended to a so-called particle system . We implemented HyperBalls using built-in Cg functions. To create the representation, it is necessary to generate envelopes using cubes and parallelepipeds to define atom and bond primitives, respectively. Then, we represent spheres and hyperboloids within these envelopes using ray casting.Starting from this previously published implementation, we were able to quickly add improvements to it, such as the lit spheres shading described below.et alText S1 in File S1. We consider implementing the whole process in a future version of UnityMol, including interactive line selection filters. For now, only some representative lines are selected according to the electrostatic potential value on the molecule surface.Electrostatic potential visualization was implemented in UnityMol either as isosurfaces or as animated particles following field lines. The latter representation is similar to the rendering described in a recent publication by Andrei Once the line coordinates are loaded into memory, we reconstruct field lines using the line renderer of Unity3D. A dash-line effect depicting moving particles with a trail is achieved using a shader that animates a sinusoidal function along the field line according to a timer. The user can change the colour, the width, the length and the speed of the particles via the GUI see  section.Molecular surfaces can be imported in Wavefront OBJ format from various molecular modelling programs such as MSMS Alternatively, UnityMol is able to generate a Gaussian molecular surface. An atomic density grid is computed by adding Gaussian distributions originating at the atom positions as discussed in All types of surfaces can be cut according to fixed or moving planes. The interior of the molecule is filled by a uniform colour to enhance the user’s impression of cutting a solid object. The implementation uses a shader hiding the pixels of the mesh located above the plane defined by the user. The colour, orientation and position of the cut plane can be changed interactively . Globally, these results depend on the size of the rendering window, and in particular concerning the HyperBalls representation, intrinsic graphics hardware performance is another key element that governs rendering performance.For entirely fluid and interactive display manipulation, it is mandatory to achieve a rendering rate above 30 Frames Per Second (FPS). Fig. S3 and Text S2 in File S1). This lead us to improve the reactivity of UnityMol by implementing an on the fly optimization as described in the following paragraph.We selected the particle system, Unity3D sphere and HyperBalls sphere methodologies to evaluate their respective efficiency limits using a set of molecules with sizes ranging from about 1 200 to 100 000 atoms see Table 1.http://www.paraview.org/) to manipulate systems with many particles interactively.To improve the overall reactivity of Unity3D, in particular for slower rendering modes such as HyperBalls spheres and bonds, we have implemented a so-called Level of Details (LoD) method. This functionality automatically switches from a specific representation that is assumed to render slowly to the fast particle system representation while the user is moving the molecule. This option is called “LoD Mode” in the UnityMol Graphical User Interface. LoD is commonly used in other generic data visualization packages such as Paraview  and when combined with the electrostatic field lines visualization. Very few molecular viewers implement this feature, which has been rendered particularly efficient and convenient to use in UnityMol.Molecular surface rendering is a fairly standard feature in molecular viewers. The addition of cut planes enables the user to easily visualize hidden elements in the interior of a molecule. This feature is particularly useful for proteins with internal cavities or channels . These functionalities can be reused for molecular visualization and interaction.There are two kinds of special effects in Unity: image based methods working in 2D screen space and methods working in 3D space. Using screen space effects, it is possible to deform an image, add black lines to outline contours or add glow and blur effects simulating persistence of vision . A visual feedback is provided by changing atom colours from white to black to represent the potential energy of each part of the elastic system.In Unity3D, a physics engine is used to model physical and mechanical behaviour of rigid 3D objects such as collision, dynamics, elastic joints, etc. … To illustrate the potential of such an engine, we have implemented an interaction with the molecule based on a very simple spring network. The user can grab atoms to deform the molecule while the structure is kept coherent  or calculate distances between atoms. The UnityMol GUI, main program features and general usage of the software are described in A particular benefit of a game engine is its modularity and the reusability of the generated routines and scripts. The use of an integrated intuitive editor facilitates the development of applications without writing numerous lines of code. It is possible to position 3D objects in the scene and apply scripts and graphical functions directly via the Unity editor. To illustrate this feature, we have adapted our molecular viewer to represent generic biological networks in 3D. To implement this network viewer (embedded in the demo), we have adapted the file parser to read XGMML (extensible Graph Markup and Modelling Language) format obtained as output of the CytoScape program We have gathered recently developed graphical tools in an easy to use distributable software that provides a unique combination of functionalities absent from other publicly available viewers. We have implemented the recently developed HyperBalls representation We compared our approach with common molecular viewers such as VMD and PyMol see . These pIn addition to molecules, we have implemented a 3D visualization of biological networks from Cytoscape Fig. S5 in File S1). This feature can be particularly useful to prototype new 3D molecular representations. Furthermore, instead of the need to recompile programs such as VMD or PyMol to see the results of modifications, using the Unity3D engine helps developers to modify, test and debug their code interactively using the editor window, on-the-fly compilation and the runtime debugger of MonoDevelop, the integrated development environment furnished with Unity3D. These features clearly help to simplify the visualization development due to a rapid and interactive trial-and-error test cycle. Furthermore, Unity3D provides simplified access to low-level graphics hardware functionalities that are usually not exposed in the APIs of existing programs such as VMD or PyMol.UnityMol is different from classical pre-compiled molecular viewer executables. In addition to the standalone and webserver versions, it can be run directly within the freely available Unity3D game engine. The game engine environment provides the user with full control over any detail of UnityMol. Because the Unity user interface is very intuitive, many simple things can be changed by the click of a button. It is straightforward to extend the application. If a GUI menu entry is missing, it can be added in minutes with just a few lines, most often by copying existing examples in the code. Moreover, when running UnityMol from within Unity3D, one can switch from game to editor mode and hand-edit the scene. Because the elements in the scene are Unity3D primitives, they can be modified on-screen by the user. For example, when visualizing field lines, the editor mode enables the user to select and delete field lines that are unwanted and hence fine-tune the scene as in Fig. 3-CThus, such a wide range of control by the user could be seen as a new paradigm for scientific software development and presents a clear rupture with current approaches used for molecular visualization tools. At best, common packages provide scripting access to selected functionality to the user, otherwise programming know-how is required to delve directly into the application source code – if it is available. In the context of open computer programs http://unity3d.com/support/community.html), which provides assistance to novice developers . In particular the existing internet forums and answer sites are a good source of information with many pieces of code almost ready-to-use. For example, the original lit sphere shader was adapted from the official Unity3D forum. Several screen-space visual effects and the file browser are user contents as well. For non-expert programmers the availability of such resources speeds-up the development cycle and softens the learning curve of the programming environment. Many common visualization techniques are already implemented and provided with Unity3D such as triangulated spheres, lines, cubes and more, as well as a physics engine and built-in functions to manage different types of controllers. While the development tools are only available on Mac and Windows, stand-alone applications can be deployed for the Linux platform as well. There are some specific limitations in the free version of the Unity3D program . Buying a professional license provides access to special features or dedicated functions for systems such as game consoles or smartphones. Work is in progress in our group to explore these additional features and deploy UnityMol on new platforms such as mobile devices.We have described how to use a game engine such as Unity3D to create a viewer for molecular structures as well as for biological networks. During the development of these tools, it became obvious that a game engine facilitates the development of scientific applications: the editor window provides an interface to position 3D objects and assist with the attribution of functionalities, e.g. implemented by a script, by dragging an icon onto the target object. Thus, the amount of lines of code to be written is rather limited and can in large parts be hidden for non-experts. Development is further facilitated by support from a very active Unity3D user community Click here for additional data file.


To find out the AMR pattern of clinical isolates of selected gram negative bacilli (GNB) to 3rd and 4th GCPs. The data were entered in WHONET & analysed& compared with each other.A total 2366 isolates  of GNB collected over 3 consecutive months were studied for AMR pattern of selected 3rd / 4thGCPs varied from 60-100 /32-100 &34-50/21-58 respectively to selected GNB, & it was significantly more among rural isolates. Among GNB, AMR was low with E.Coli & Klebsiella but high among Pseudomonas.In rural & urban isolates the AMR pattern for 3A heterogeneous AMR pattern observed in the study was comparable with published reports. The probable reasons for wide variations were due to overuse, misuse& dysregulated availability of antimicrobials; promotional incentives & high profit margins; & demands of the community. These push the rural practitioners to use newer generation of antimicrobials frequently. Also lack of adherence to medication, suboptimal regulatory system , lack of prescribing policies and to some extent technical aspects of GNB, might have contributed to increased AMR in rural areas. Hence there is an urgent need to design & implement antimicrobial policy & surveillance system at regional & institutional level under national guidelines.None declared.


Evidence links dopamine release in the mid-brain to the pathophysiology of psychosis, addiction and reward. Repeated ingestion of refined carbohydrate may stimulate the same mesolimbic dopaminergic pathway, rewarding such eating behaviour and resulting in excessive food intake along with obesity. In this paper, we explore the role of dopamine in reward and psychosis, and discuss how reward pathways may contribute to the weight gain that commonly follows antipsychotic drug use, in people with psychotic illness. Our theory also explains the frequent co-occurrence of substance abuse and psychosis. From our hypothesis, we discuss the use of carbohydrate modified diets as an adjunctive treatment for people with psychosis. The mesolimbic dopaminergic system of the mid brain is intimately linked to two major categories of disease 1) addiction, and 2) psychotic disorders. Although, the link between mental illness and addiction has been commonly reported [ addictio psychotiTo explore the intersection of psychotic and addiction phenomena, first, we briefly define addiction, along with its clinical features. Next, we review the neuroanatomy and physiology of the dopaminergic pathways in the midbrain, and how this region behaves in both disorders. We then discuss the mechanism of action of antipsychotic drugs and new evidence linking food intake and obesity with addiction. Our hypothesis then links psychosis, food and addiction and we explain how this may offer new dietary therapy for people with schizophrenia.repeated failures to refrain from drug use despite prior resolutions to do so” [When does a person’s substance use cross the boundary to become an addiction? Controversy surrounds the definition, with different conceptions offered. Heather describes addiction as “o do so” . The DSMo do so”  criteriaThe last feature of the DSM-IV criteria, the withdrawal syndrome, may be the most important, given the subjective nature of the other features. Withdrawal symptoms include depressed mood, disturbed sleep and a wide variety of somatic symptoms  and may In the last thirty years the origin of clinical symptoms of addiction has been linked to a part of the brain responsible for subconscious behaviour and motivation, and a specific neurotransmitter: dopamine. The dopaminergic system is composed of two major pathways: (1) the nigrostriatal system that projects from the substantia nigra in the basal ganglia to the corpus striatum and (2) the mesocorticolimbic dopamine system, which emanates from the ventral tegmental area of the mid brain to the nucleus accumbens and olfactory tubercle , medial pre-frontal cortex and amygdale  . These nAlso, extracellular dopamine release in the striatum (from psychostimulants) and subjectively feeling “high” or euphoric  are closThe nigrostriatal pathway, which includes the substantia nigra, is best known for its relationship to the degenerative disorder, Parkinson’s disease, characterised by a cluster of motor and non-motor symptoms. Motor symptoms include a characteristic ‘pill-rolling’ tremor, shuffling gait, brady-kinesia (slowing of motor movements), and rigidity. Non-motor manifestations consist of depressed mood, cognitive impairment along with autonomic and sleep disturbances. Replacement of dopamine, in precursor form (levodopa), partially relieves many of these symptoms. Degeneration of the dopaminergic neurones in this part of the brain are seen in post mortem studies of people who have died with Parkinson’s disease .The mesolimbic dopaminergic pathway is activated by drugs and substances that people ingest, inject, snort or smoke that, ultimately, cross the blood brain barrier, after entry into the circulation. Over the last two decades, other evidence has implicated this pathway in weight control and eating behaviour. This evidence comes from a number of different studies. These include microdialysis, rodent based research of the mesolimbic pathway that have shown that exposure to food results in dopamine release. Although the quantity is less than what occurs in response to drugs of abuse, such as amphetamines . The magIt is also recognised that the brain’s primary fuel in the non-fasting state is glucose, and changes in venous glucose concentrations trigger changes in the firing of dopaminergic neurones in the striatum . Other sTolerance or need for an increased quantity of a substance to get the same ‘hit’ is a dominant feature of addiction and may reflect reduced sensitivity to dopamine by post-synaptic neurones in the nucleus accumbens. Anatomical changes found following Positron Emission Tomography studies of those who suffer from drug addiction show evidence of adaptation, with an increased concentration of dopamine receptors compared to controls . Such chOf all the food groups, carbohydrate is commonly ascribed addictive properties , and witvia a gastric fistula - concentration-dependent dopamine release follows [The mechanism by which sucrose induces dopamine release is uncertain, however, two possibilities are commonly cited . First,  follows . Further follows . Such fiWhat are the clinical features of drug induced dopamine release in human subjects? Two principal effects are commonly described . First, As well as playing a role in addiction, dopamine has been referred to as the “wind of the psychotic fire”, when describing its association with the pathophysiology of schizophrenia . Evidenc2 receptors [2A is a feature of atypical antipsychotic agents, such as clozapine and olanzapine. Although dopaminergic dysfunction is highlighted most frequently in the pathophysiology of psychosis, other neural paths are also implicated, such as the glutaminergic and serotonergic [Antipsychotic or ‘neuroleptic’ drugs have a specific mechanism of action. After entry into the circulation they diffuse across the blood-brain barrier. There, they bind to and block neural circuits, specifically interfering with the action of dopamine on mesolimbic and mesocortical Deceptors . Greatertonergic  ones.Given the association between dopaminergic neural paths and addiction and psychosis, at a clinical level we may expect a close relationship between the two disorders. For example, in one summary, the prevalence of smoking was between 80 and 90% in people treated in hospital with schizophrenia . Numerou2 receptor [2 receptor in vitro correlated to therapeutic free neuroleptic levels [2 receptor site  along with increased serotonergic effects.Researchers have concluded that no drug with significant antipsychotic action has been identified that does not have a significant affinity for the Dreceptor . In the c levels . The altFor a long time, activity in the dopaminergic system has been difficult to directly assess in human subjects, but recently, positron and photon emission tomographic techniques have enabled this pathway to be studied in detail. Several connections between dopamine and psychosis are described. First, amphetamine induced dopamine release is exaggerated in people with schizophrenia; second, after amphetamine administration, dopamine release is associated with psychotic symptoms; and third, these changes in dopaminergic physiology in people with schizophrenia have been detected in patients never treated with antipsychotic drugs . This laWhat is the consequence of excess ‘reward’ or ‘re-inforcement’ from dopamine release? Recent theories suggest that in people with schizophrenia, dopamine release leads to aberrant salience or focussing on innocuous stimuli . SymptomIf dopamine is the principal neurotransmitter mediating both re-enforcing behaviour and reward from ingestion of food, what are the likely consequences of blocking this action with antipsychotic drugs? Our hypothesis links the increased prevalence of obesity found in people treated with anti-psychotic agents and the known pharmacodynamic properties of such drugs - blocking dopamine effects in the reward centre of the mid-brain. We speculate that to compensate for the reduced reward after taking neuroleptics, patients exaggerate the stimulation of this pathway by increasing their food intake, or use of other addictive substances.2) receptor agonist [Rodent studies have supported our hypothesis that antipsychotic drug use enhances feeding behaviour. In general, long term exposure of rodents to antipsychotic agents is associated with weight gain , althoug agonist . Four an agonist .In human subjects, weight gain commonly occurs in association with antipsychotic use, however the mechanism underlying this effect is not well understood. Several explanations are proposed, including increased appetite, leading to excess energy intake . MetabolWe speculate that if dopaminergic neural transmission is blocked by antipsychotic drugs in patients with schizophrenia, compensation may occur, with patients adapting by carrying out behaviours that stimulate dopamine release (from food or drug intake) in an effort to maintain homeostasis, achieving the same feeling of reward, satisfying hunger or drug withdrawal symptoms. Evidence for such an effect can be found in animal experiments which demonstrate dulling effects of antipsychotic drugs on chemical or electrical stimulation of mid- brain reward paths . Also, a2 receptors, they vary substantially in the amount of serotonergic, histaminergic and adrenergic activity they exhibit. Such a finding may indicate that other pathways or neurotransmitters are more likely to be causally implicated in weight gain side effects than the dopaminergic path. However, in studies which observe weight gain in patients treated for more than one year, such differences between drugs are less marked, and some of the between drug heterogeneity may be explained by between-study variation in dosing [Our theory focuses on the centrality of the dopamine receptor blockade of antipsychotic drugs and their simultaneous effects on weight gain and psychosis. One potential flaw in this theory is the differential effects on weight gain of different drugs. For example, in studies in which patients are followed for less than one year of treatment, a clear hierarchy of weight gain liability occurs, with clozapine>olanzapine>risperidone>ziprasidone=aripiprazole . Althougn dosing .How might our theory improve care for people with psychosis? We speculate that if addiction and psychosis share common features and a common neuroanatomical pathway, with dopamine release and disordered reward and motivation, a key treatment for reducing the severity of psychosis may involve reducing stimulation of the mid brain. If antipsychotic drugs exert their effect, principally, by blocking the effects of mesolimbic dopamine release, we expect that reduced release of dopamine in this pathway may result in similar beneficial effects on psychotic symptoms in people affected by schizophrenia. Although still controversial, we speculate that food may be a key stimulant of this disordered pathway, and altering diet may improve psychosis and reduce the need for antipsychotic treatment. If blocking the effects of free dopamine reduces psychotic symptoms, then reducing dopamine release is likely to induce a similar effect. Offering treatment for other addictions (not only food) to limit dopamine release also follows from our theory.Surprisingly, evidence for nutrition interventions to improve psychotic symptoms has received little attention. Peet speculated that the co-occurrence of schizophrenia and diabetes, even in drug naive patients, suggests that the two disorders share a common aetiology . One stuIf a dietary approach to treating psychotic symptoms was suggested, what might this regime look like? Glycemic index (GI) describes the rate of absorption of glucose from carbohydrate. High GI carbohydrate foods, such as white bread, sugar and refined cereals, cause a ‘spike’ in insulin and blood glucose (followed by a dip). Low GI carbohydrate foods, such as wholegrain bread and most vegetables , produce a slower insulin response and thus a more steady supply of glucose to the blood. Low GI diets induce weight loss and improve blood glucose control in people with diabetes. A recent meta-analysis showed that people following a low GI diet had more weight loss than other diets . Other mStudies that measure the effect of carbohydrate modified diets on psychotic symptoms would test whether our hypothesis is tenable. Drop out and compliance with carbohydrate restricted or modified diets is often low, and engagement of people with schizophrenia in such treatment may be more complicated than those which involve obese people. Nevertheless, a non-pharmacological approach to the treatment of psychosis may be more acceptable than drug treatments that are often associated with unpleasant side effects and low levels of adherence to treatment. Carbohydrate modified diets may also provide an adjunct to antipsychotic medication, potentially limiting unintended effects such as weight gain and adverse increases of other indices of cardiovascular risk. Meta-analyses of low glycaemic index diets show that these interventions are effective for achieving sustained weight loss in obese people  and reduOur hypothesis also reinforces the rationale for treatment of addictions in people with schizophrenia. The use of short-term nicotine replacement therapy and quit advice, limitation of caffeine intake, and treatment of methamphetamine, alcohol and other substance abuse in such patients are likely to not only improve the patient’s general health, but also assist their recovery from psychosis.Our theory provides a parsimonious and testable hypothesis, linking the action of antipsychotic agents with commonly reported side effects. It also explains the common co-occurrence of schizophrenia with addiction, obesity and diabetes. The common link drawn between eating, psychosis and mid-brain dopaminergic reward, logically, suggests that psychosis may be improved, by modifying carbohydrate consumption. We consider that such an idea should be tested in clinical trials.


Unixenus corringlensis Short & Huynh, sp. n., the latitude given for the single collecting event of this species in Corringle state forest is incorrect and should be 33°40'00"S not 33°22'12"S.When describing the new species The correct wording in the species description for the holotype, a male paratype and two specimens included as other material is as follows:33°40'00"S, 147°15'00"E (±1 km) , 21–26 March 1996, collected by D. Smith, AM KS.119541. Specimen mounted on slides,deposited in AM.Holotype. Female, Corringle State Forest, NSW, Paratypes. 1 male, same collection as holotype, AM KS.119540.Other material. 2 immature specimens, (sex and stadia not determined), same collection as holotype, AM KS.53604, preserved in ethanol.The Table of localities included as an appendix also requires the same correction. Portion of the table is shown below with corrections highlighted in yellow.


OGD was achieved by reducing the aCSF glucose concentration and by bubbling with 95%N2/5%CO2. After approximately 5 min OGD a large dopamine efflux was observed, presumably caused by anoxic depolarisation. On applying a second OGD event, 60 min later, dopamine efflux was delayed and reduced. We first examined the effect of varying the length of the conditioning event from 5 to 40 min and found tolerance to PIC increased with increasing duration of conditioning. We then examined the receptor mechanism(s) underlying PIC. We found that pre-incubation with either MK-801 or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) reduced tolerance to the second OGD event. These data suggest that either N-methyl-d-aspartate (NMDA) or adenosine A1 receptor activation evokes PIC in the mouse caudate.Evidence suggests that pre-ischeamic conditioning (PIC) offers protection against a subsequent ischeamic event. Although some brain areas such as the hippocampus have received much attention, the receptor mechanisms of PIC in other brain regions are unknown. We have previously shown that 10 min oxygen and glucose deprivation (OGD) evokes tolerance to a second OGD event in the caudate. Here we further examine the effect of length of conditioning event on the second OGD event. Caudate mouse brain slices were superfused with artificial cerebro-spinal fluid (aCSF) bubbled with 95%O However, in other brain areas, other neurotransmitters, including dopamine, contribute to ischeamic damage. Massive release of dopamine occurs in the caudate in response to anoxia  is an endogenous neuroprotective mechanism by which a sub-lethal conditioning event provides tolerance to a subsequent ischeamic event . We havein vitro  and in vin vitro , and maychannels , to bringnalling , reducin1 or glutamate NMDA receptor signalling affects PIC.The present study used fast cyclic voltammetry in mouse caudate slices to further examine the effect of PIC on OGD-evoked dopamine release. Dopamine efflux after OGD in rodent caudate slices has been shown to be very similar to that seen in vivo after cardiac arrest . We aime22.1Adult male mice  kept on a 12/12 h light dark cycle with freely available food and water were killed by cervical dislocation with no anaesthesia, an approved schedule 1 method. Anaesthetics are neuroprotective in this model .2.22/5% CO2, at room temperature (21±1 °C). The slices were equilibrated in the slice saver to allow recovery from trauma associated with slicing  and continuously superfused with aCSF (250 ml/h) for 45 min. The perfusion medium was then switched to OGD aCSF for 0–40 min depending on the experiment (see later) after which slices were superfused for a further 60 min in aCSF and then exposed to a second OGD event.Maintenance aCSF (mM): NaCl (126.0), KCl (2.0), KH2PO4 (1.4), MgSO4 (2.0), NaHCO3 (26.0), CaCl2 (2.4), (+)-glucose (10.0), bubbled for at least 60 min with 95% O2/5% CO2. OGD aCSF (mM): NaCl (1 2 6), KCl (2.0), KH2PO4 (1.4), MgSO4 (2.0), NaHCO3 (26.0), CaCl2 (2.4), (+)-glucose (2.0), bubbled for at least 60 min with 95% N2/5% CO2.The composition of aCSF was 2.3Extracellular dopamine concentrations in the dorso-lateral caudate nucleus were measured by FCV at carbon fibre microelectrodes. Carbon electrodes were made by inserting an 8 μm diameter carbon fibre into a 10 cm length borosilicate glass capillary , which was pulled using an PE21 electrode puller , such that the carbon fibre protruded from the pulled tip. The carbon fibre was then cut to a length of 75 μm.A stainless steel auxiliary electrode and a Ag/AgCl reference electrode were placed in the slice chamber remote from the slice. Voltammetric scans  were applied at 1 Hz using a Millar voltammeter . Under these conditions dopamine oxidised at +600 mV and reduced at −200 mV . VoltammδDA/δt) as previously described  the slice can spontaneously release large amounts of dopamine . Perfusiescribed .2.42.4.1After 45 min equilibration a slice was exposed to 0, 5, 10, 15, 20, 30 or 40 min OGD. Thereafter the slice was exposed to 60 min of oxygenated aCSF and then exposed to a second OGD event of at least 20 min duration. Thus we could examine the effect PIC (0–40 min) on the second OGD event 60 min later .2.4.21 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine  and of the NMDA receptor antagonist, MK-801 (10 μM) on the tolerance evoked by 10 min PIC. The slice was superfused with aCSF containing either DPCPX or MK-801 for 45 min. The superfusate was then changed to OGD aCSF containing the respective drug, for 10 min, after which the superfusion aCSF was switched to drug-free aCSF for 60 min, then to drug-free OGD for at least 20 min. We used a second OGD event of 20 min or more (vs 10 min for the conditioning event) because in preliminary experiments the OGD-evoked dopamine efflux was found to be delayed to such an extent that on some occasions 10 min OGD is not long enough to evoke dopamine efflux.We measured the effect of the adenosine-AOn completion of these protocols some slices were removed from the slice chamber and prepared for TTC staining to assess mitochondrial function, or for HPLC analysis of tissue content of dopamine, as described below.2.5After OGD superfusion, slices were incubated in aCSF at room temperature (21±1 °C) for 30 min, then transferred into sterile cell culture wells and stained with TTC (2%w/v in 0.9% NaCl) for 30 min in the absence of light. The slices were then fixed with 4% paraformaldehyde solution, photographed and the percentage of the caudate area stained was assessed using Image software .2.62PO4, 1.2 mM octane sulphonic acid, 1.0 mM EDTA and 10% methanol, pH 3.7. The mobile phase was delivered at 110 μl/min, using a Jasco high pressure pump. Electrochemical detection was at a glassy carbon working electrode, set at 700 mV relative to Ag/AgCl, using an Antec Intro electrochemical detector incorporating a VT-03 low volume flow cell (Antec Instruments). The concentration of dopamine was measured relative to standard solutions (100 nM), using ChromPerfect data analysis software (Justice Laboratory). Each sample was run in duplicate, and the mean tissue content of dopamine was calculated and normalised to the wet weight of tissue in nM/mg.On termination of FCV experiments, slices for HPLC analysis were removed from the chamber and the caudate dissected out on ice, placed in a microcentrifuge tube and weighed. Perchloric acid  was added to the tube and the tissue was homogenised for 20 s . The homogenate was centrifuged , and the supernatant taken for HPLC analysis. A 15 μl aliquot of sample was injected automatically and separation was achieved on a C18 Luna reverse phase column , with a mobile phase comprising 75.0 mM NaH2.7All chemicals were purchased from Sigma Aldrich. DPCPX was initially dissolved in DSMO to make a stock solution of 10 mM, and then diluted in aCSF to give a working concentration of 5 μM, a concentration that has previously been shown to attenuate anoxia-evoked PIC in hippocampal brain slice preparations . MK-801 2.8P<0.05 was considered significant. SigmaStat software was used for all statistical analysis. Values are means±SEM.Where the effects of one condition were to be compared with another, a two-tailed, unpaired student t-test was done. When there were more than two conditions with one variable, a one-way analysis of variance (ANOVA) was done with post hoc analysis with Tukey's post test for multiple comparisons. A two-way ANOVA was done when two independent factors were present. A probability of 3Consistent OGD-evoked dopamine release occurred 350±82 s after the onset of OGD, taking 148±17 s to achieve peak-dopamine efflux of 20.3±4.0 μM. The rate of change of dopamine efflux was 73±19 nM/s .3.1F=46.88, P<0.05); F=3.867, P<0.05) but the other PIC events (10–40 min) had no effect on T-peak. All PIC events (5–40 min) significantly reduced the OGD-evoked peak-dopamine, from 20.3±4.0 μM in control slices to 3.6±1.1 μM 60 min after 40 min PIC, F=7.58, P<0.05. All PIC events (5–40 min) significantly reduced δDA/δt, from 73 nM/s in control slices to 19 nM/s 60 min after 40 min PIC =4.39, P<0.05; Single PIC events of 5–40 min evoked a significant delay in T-on of OGD-evoked dopamine release 60 min later  much was released on the first OGD event or (2) the slices' ability to synthesise dopamine was now compromised. We therefore examined caudate dopamine tissue content immediately after the first OGD event and, in different slices, after 60 min incubation following OGD. The data show that dopamine tissue content is reduced after OGD, but that it recovers to near control levels after 60 min reperfusion A.F=29.95, P<0.05) and a PIC time X incubation interaction =5.79, P<0.05). Tukey's test revealed that, immediately after the PIC event, all three PIC treatments  had reduced the dopamine content vs no PIC . However, after 60 min reperfusion dopamine content partially recovered in that now only the 20 and 30 min PIC was different from controls  while in the 30 min PIC group dopamine content increased over the 60 min reperfusion (P<0.05) and there was a tendency for dopamine content to increase after reperfusion in the 10 min PIC group (P=0.057). These data suggest that even after 30 min of OGD the slice can recover its dopamine synthesising capacity.There was a main effect of PIC time =6.67, P<0.05) and Tukey's test revealed that there was a loss of TTC staining after 20 min OGD vs both 0 and 10 min OGD =37.43, P<0.05). There was no main effect of MK-801 =0.01, P=0.93) but there was a PIC × MK-801 interaction =16.12, P<0.05). Post hoc analysis revealed that PIC increased T-on in the absence of MK-801 (P<0.05), but there was no PIC-induced increase in T-on in the presence of MK-801 (P=0.18). MK-801 also increased T-on in the absence of PIC (P<0.05). Thus, MK-801 increases T-on in caudate slices that have not been conditioned, but attenuates the increase in T-on after PIC (We superfused the caudate slice with MK-801 for 45 min prior to 10 min OGD conditioning (with MK-801 present during PIC). We then subjected the slice to a second OGD event 60 min later and measured the dopamine efflux parameters on the second OGD-evoked dopamine release event. PIC increased T-on after OGD =2.48, P=0.13), nor did PIC =0.5, P=0.49) and there was no PIC × MK-801 interaction =0.93, P=0.34). PIC did reduce peak-dopamine =30.6, P<0.05) but MK-801 had no effect on peak-dopamine =0.069, P=0.89) and there was no PIC × MK-801 interaction =0.14, P=0.71). PIC decreased δDA/δt =31.8, P<0.05) as did MK-801 =16.5, P<0.05) and there was a PIC X MK-801 interaction =9.7, P<0.05). Thus MK-801 slowed down the rate of dopamine efflux in slices which had not been exposed to PIC, but had no effect in slices after PIC =7.69, P<0.05). Thus PIC increased T-on (p<0.05) and DPCPX reduced the PIC-induced increase in T-on (P<0.05). In the absence of PIC, DPCPX had no effect on T-on (P>0.3) and there was no effect of DPCPX on δDA/δt (P>0.7) or on peak-dopamine . We then subjected the slice to a second OGD event 60 min later and measured the dopamine efflux parameters on the second OGD-evoked dopamine release event. ANOVA revealed a PIC X DPCPX interaction =0.88, P=0.36), however, MK-801 reduced the OGD-evoked loss of TTC staining =89.5, P<0.05). There was no interaction between PIC and MK-801 =0.01, P=0.91). Thus MK-801 attenuated the loss of TTC staining regardless of whether the slice had been exposed to PIC or not =2.55, P=0.13) and no interaction between PIC and DPCPX =1.18, P=0.29; There was no significant effect of PIC on caudate TTC staining immediately after the final OGD event  adenosine A1 receptors were not sufficiently blocked or (2) the delay in T-on found is mediated through additional mechanisms e.g. the NMDA receptor. Even though the delay in T-on was not fully attenuated the results suggest that during PIC, activation of adenosine A1 receptors is involved in the cellular pathway responsible for the tolerance to an OGD insult in the caudate.We have found MK-801 to attenuate PIC in the caudate. Blockade of PIC by NMDA receptor antagonists has been achieved in vivo in the hippocampus  and in v delayed  and rapi delayed  latency f models . Since iThere is an apparent mismatch between our voltammetry and TTC data in that PIC evokes tolerance to a second OGD event but no apparent tolerance to toxicity, as determined by TTC staining . Thus al51 receptor antagonist, DPCPX or the glutamate-NMDA receptor antagonist, MK-801. Given that adenosine A1 and NMDA receptors are likely to be involved in PIC; these could be future targets to induce neuroprotection pharmacologically. These data, involving OGD-evoked dopamine efflux in the caudate, add to the wealth of data on PIC from the hippocampus and cortex and suggest receptor mechanisms that may be involved in PIC throughout the brain.Attenuating or slowing ischaemia-evoked dopamine efflux may limit neuronal damage after stroke. We have shown that brief exposure to OGD in vitro elicits rapid adaptive mechanisms that delay the onset of dopamine efflux during a secondary OGD episode. We propose that increases in T-on and T-peak are indicative of neuroprotection as the brain is more tolerant to subsequent OGD. Our main finding was that the tolerance achieved with 10 min conditioning was attenuated in the caudate by the application of either the adenosine A


Oxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER) and nucleotide incision repair (NIR). Hydantoin derivatives such as 5-hydroxyhydantoin (5OH-Hyd) and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd), major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells.in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts.Here, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 5′ next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vivo.This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions  Endogenous aerobic metabolism and variety of exogenous factors generate reactive oxygen species (ROS), which can damage macromolecules including lipids, proteins and nucleic acids. DNA has a limited chemical stability and it is one of the most biologically critical targets for ROS, with more than 80 base modifications identified so far E. coli Xth protein +3, Pb+2, Ca+2, Mn+2 and Mg+2+2 cations can support APE1-catalyzed NIR activity in human cell-free extracts in vivo.In human cells the major AP endonuclease 1, APE1/Ref-1/HAP-1, initiates NIR pathway by cleaving duplex DNA 5′ next to oxidatively damaged bases 5-C6 double bond, generating thymine and uracil glycols, 5,6-dihydro-5,6-dihydroxythymine and 5,6-dihydroxy-5,6-dihydro-uracil, respectively. Hydantoin, also known as glycolyurea, derivatives: 5-hydroxyhydantoin (5OH-Hyd) and 5-hydroxy-5-methylhydantoin (5OH-5Me-Hyd)   have beeE. coli Nth, Nei and Fpg as well as S. cerevisiae Ntg1 and 2 can initiate the BER pathway for 5OH-Hyd and 5OH-5Me-Hyd residues in DNA E. coli Nfo, yeast Apn1 and human APE1 initiate the NIR pathway by incising the duplex DNA containing 5OH-Hyd and 5OH-5Me-Hyd residues. Using Matrix Assisted Laser Desorption Ionisation Time-Of-Flight (MALDI-TOF) Mass Spectrometry (MS) analysis we show that the AP endonucleases cleave 5′ next to the pyrimidine-derived hydantoin lesions when present in the duplex DNA substrate. The removal of 5OH-Hyd and 5OH-5Me-Hyd residues in the both BER and NIR pathways in bacteria, yeast and human cells has been characterized. A number of redundant DNA glycosylase activities excising the pyrimidine-derived hydantoins have been identified, including human NTH1, NEIL1 and NEIL2 DNA glycosylases. The roles of BER and NIR, as back-up complementary pathways for oxidized DNA bases in vivo, are discussed.Previously, it has been shown that E. coli and S. cerevisiae bifunctional DNA glycosylases initiate BER pathway by excising 5OH-Hyd and 5OH-5Me-Hyd residues when present in duplex DNA 32P]-labelled 5OH-Hyd•G and 5OH-5Me-Hyd•A substrates with the E. coli Nfo, S. cerevisiae Apn1 and human APE1 proteins. As shown in versus 6) 32P]-labelled 14-mer size marker oligonucleotides containing either cytosine or thymine at 5′ end , suggesting that either cleavage fragments with 5′-terminal nucleotide migrate faster as compared to 14-mer size marker or that 5OH-Hyd may undergo some transformation after enzymatic cleavage.versus 4,5,7). Again, APE1 cleaved 5OH-5Me-Hyd•A only under NIR conditions generating the cleavage product with the same size as with 5OH-Hyd•G oligonucleotide (lanes 15 and 5). In contrast, Nfo and Apn1-catalyzed incision resulted in two closely migrating (n+1) fragments (lanes 14 and 17), upper-band co-migrates with 14-mer size marker (lane 18) and lower-band co-migrates with 5OH-Hyd•G NIR-cleavage fragment . Taken together, these results suggest that the Apn1 and Nfo-generated upper-band corresponds to the fragment containing a 5′-dangling 5OH-5Me-Hyd nucleotide whereas the lower-band is similar to that observed with 5OH-Hyd•G substrate and may correspond to fragment containing a decomposed form of 5OH-5Me-Hyd. We may propose that bigger molecular weight difference between 5OH-5Me-Hyd and its putative degradation product resulted in a better electrophoretic resolution on the gel as compared to 5OH-Hyd and its corresponding putative degradation product. Importantly, Nfo-catalyzed cleavage of 5OH-5Me-Hyd•A under NIR condition gives essentially the same proportion of two closely migrated bands on the gel as compared to the Nfos' standard reaction condition. Furthermore, longer incubations and/or simultaneous treatment of 5OH-5Me-Hyd•A with Nfo and APE1 do not change the migration pattern of cleavage products , and the other one with molecular mass [M-H]− = 3953 Da corresponding to a 13-mer oligonucleotide 5′-p-ZTGTGACTGATCC  is decomposed to an ureido residue (Z of 59 Da). In addition to the cleavage products we can see the presence of the mono- and bi-charged peaks corresponding to the complementary strand . Importantly, the respective amounts of cleavage fragments containing ureido and 5OH-Hyd varied among the experiments. As shown in XTGTGACTGATCC with [M-H]− = 4023 Da which closely corresponds to calculated [M-H]− = 4023.5 Da and an additional peak with [M-H]− = 3953.5 Da corresponding to the 13-mer 5′-p-ZTGTGACTGATCC that contains an ureido residue. As expected the MALDI-TOF MS analysis did not reveal any cleavage of the complementary strands   . It shou 6260 Da . This reE. coli Fpg and Nei proteins, NEIL1 is a bi-functional DNA glycosylase endowed with an AP lyase activity that incises DNA at abasic sites by a β,δ-elimination mechanism and leaves single-strand DNA break carrying a phosphate residue at the 3′ and 5′-termini . As expected, MALDI-TOF MS analysis revealed two mono-charged peaks one at [M-H]− = 2776.8 Da corresponding to the 9-mer oligonucleotide released 5′ upstream to the lesion 5′-CACTTCGGAp  and the other one at [M-H]− = 3714.6 Da corresponding to the 12-mer oligonucleotide released 3′ downstream to the lesion 5′-pTGTGACTGATCC   . This re 3715.5) .32P]-labelled Tg-34•A and Tg-30•A oligonucleotide duplexes but not 19Tg-IW•A duplex (Supporting Information E. coli exonuclease III (Xth) protein Thymine glycol is a major nucleobase lesion that may be formed within DNA by several oxidative processes. This oxidized form of pyrimidine residue, as hydantoins residues, exhibit a ring-chain tautomerism at C6-N1 or C5-N1 bond that may lead to degradation towards fragmented products (such as ureido or formamido residues), spontaneously and/or upon oxidative and alkali conditions E. coli and yeast. Here, we investigated whether 5OH-Hyd and 5OH-5Me-Hyd residues are also substrates for the previously characterized bacterial and human DNA glycosylases. As shown in 32P]-labeled 5OH-Hyd•G and 5OH-5Me-Hyd•A oligonucleotide duplexes were challenged with a variety of highly purified DNA glycosylases. Since not all DNA glycosylases possess AP site-nicking activity, the samples, after incubation with the mono-functional DNA glycosylases, were treated with light piperidine treatment [10% (v/v) piperidine at 37°C for 45 min]. in order to cleave DNA at the potential abasic sites generated by the excision of the modified base. Control light piperidine treatment of 5OH-Hyd•G duplex resulted in slight defragmentation of the oligonucleotide to short DNA fragments indicating the presence of alkaline labile sites most likely due to spontaneous depurination . Interestingly, TDG, a human homologue of MUG, does not show any detectable activity on the hydantoins (lane 6). Overall, the E. coli Nei and Nth proteins show a slight preference for 5OH-5Me-Hyd than 5OH-Hyd , whereas human NEIL1 and to more extent NEIL2, NTH1 and E. coli Fpg excise preferentially 5OH-Hyd than 5OH-5Me-Hyd residues , DNA polymerase β (POLβ) and DNA ligase I (LIG1) 2 which allowed us to bring the concentration of free Mg2+ down to 1 mM, due to binding of magnesium cations by ATP. Here, we examined whether 5OH-Hyd residues can be efficiently removed in the APE1-initiated NIR pathway in vitro in DNA glycosylase-independent manner (2 and 2 mM ATP) which allows both NIR endonuclease activity and DNA repair synthesis, APE1 incised about 60% of 3′-[32P]-end labelled 5OH-Hyd•G substrate resulting in a 14-mer cleavage product (lane 2). Addition of FEN1 initiates strong 5′→3′ exonuclease degradation of 14-mer fragment (lane 4). Previously, we observed futile repair of αdA•T in the presence of APE1 and LIG1 proteins Recently, we have reconstituted t manner . Under tin vivo, we examined AP endonuclease and DNA glycosylase activities in cell-free extracts from E. coli and human cells. To distinguish NIR and BER activities in the extracts we used 3′-[32P]-labelled 5OH-Hyd•G and 5OH-5Me-Hyd•A oligonucleotide duplexes as substrates. When using these substrates the DNA glycosylases/AP lyases generate a 13-mer cleavage fragment . As expected from the known induction of the Nfo protein by paraquat, addition of this oxidizing agent to growing cultures of E. coli increased the amount of the 14-mer fragment (lanes 3 and 15). Also, extracts from E. coli nfo mutant completely lack NIR activity on both DNA substrates (lanes 5 and 17) indicating that the NIR pathway in E. coli is absolutely dependent on the Nfo gene product. Interestingly, extracts from the E. coli nth fpg and nth nei double mutants exhibited dramatic decrease in incision activities on both 5OH-Hyd•G and 5OH-5Me-Hyd•A substrates  as compared to the extracts from E. coli WT, single nth, nfo and double fpg nei mutants (lanes 2–6 and 14–18). Importantly, in the extract from E. coli nth nei double mutant we detected only NIR activity and no DNA glycosylase (lanes 8 and 20) suggesting that Nth and Nei are major DNA glycosylases that remove 5OH-Hyd and 5OH-5Me-Hyd residues in the BER pathway. Taken together, these results suggest that in E. coli pyrimidine-derived hydantoins are mainly removed in DNA glycosylase-initiated BER pathway. However, in the absence of DNA glycosylases, the NIR pathway can serve as back-up system.As shown in nth mutant on 5OH-Hyd•G  2+ and BER+EDTA (in the presence of 1 mM EDTA and absence of Zn2+ cations that may inhibit the DNA glycosylases) conditions  and 5-hydroxycytosine (5OHC) containing DNA duplexes under NIR condition . This suggests that either 3′-P residues are removed by a 3′-phosphatase activity or that NTH1 could be a major hydantoin-DNA glycosylase activity in human cell extracts. To test this hypothesis we down-regulated NTH1 expression using specific siRNA duplex 32P]-labelled DHU•G oligonucleotide duplex as a substrate  cells and FA-C cells complemented with a plasmid expressing FANCC protein (FA-C+FANCC). FA-C extracts exhibit somewhat decreased DNA glycosylase activity on 5OH-Hyd•G as compared to AHH1 and FA-C+FANCC extracts suggesting that FA-C cells are impaired in the repair of 5OH-Hyd residues (Supporting Information Fanconi anemia (FA) is a recessive cancer prone syndrome featuring bone marrow failure, hypersensitivity to interstrand DNA cross-links (ICLs) and also to ionizing radiation and oxidative stress Oxidized bases are the major endogenous DNA lesions that can accumulate during aging. Purine and pyrimidine moieties of the respective nucleosides undergo oxidative degradation, resulting in a number of modified bases that can be highly mutagenic when present in DNA. 5OH-Hyd and 5OH-5Me-Hyd residues have been shown to be major oxidation decomposition products of cytosine and thymine, respectively i) incise the oligonucleotide duplexes 5′ next to 5OH-Hyd and 5OH-5Me-Hyd residues generating 3′ downstream cleavage fragments still containing 5′-terminal damaged nucleotide; (ii) degrade 5′ upstream cleavage fragments by their non-specific 3′→5′ exonuclease activity  since under this condition APE1 cannot bind to DNA substrate in the tight manner and catalyze the NIR activity  conditions, three human DNA glycosylases can excise 5OH-Hyd and 5OH-5Me-Hyd residues hence contributing to the redundancy in DNA repair pathways that may back-up each other and/or act preferably depending on chromatin context, DNA damage signalling pathway and various cellular regulation mechanisms. Study of the BER activities in HeLa cell extracts demonstrated that NTH1 is a major detectable DNA glycosylase activity towards 5OH-Hyd and 5OH-5Me-Hyd residues in DNA  (PerkinElmer), or 3′-end labelled by terminal deoxynucleotidyl transferase (New England Biolabs) in the presence of [α-32P]-3′-dATP  (PerkinElmer) as recommended by the manufacturers. Radioactively labelled oligonucleotides were desalted with a Sephadex G-25 column equilibrated in water and then annealed with corresponding complementary strands for 3 min at 65°C in a buffer containing 20 mM HEPES-KOH (pH 7.6), 50 mM KCl. The resulting duplex oligonucleotides are referred to as X•G , respectively, where X is a modified residue.Sequences of the oligonucleotides used in the present work are shown in The siRNAs sequences used to decrease APE1 and NTH1 in HeLa cells have been taken from previously described studies 32P-labelled 5OH-Hyd•G and 5OH-5Me-Hyd•A oligonucleotide duplexes. Assays for the DNA glycosylases activities were performed in a “BER+EDTA” buffer containing 50 mM KCl, 20 mM HEPES-KOH (pH 7.6), 0.1 mg·mL−1 BSA, 1 mM DTT and 1 mM EDTA and 2.5 nM of a pure protein, unless otherwise stated. The release of 5OH-Hyd and 5OH-5Me-Hyd base adducts was measured by the cleavage of the oligonucleotide containing a single lesion at a defined position. For the monofunctional DNA glycosylases, the abasic sites left after damaged base excision action were cleaved by light piperidine treatment (10% (v/v) piperidine at 37°C for 10 min).The standard reaction mixture (20 µL) contained 5 nM of M  were incubated under respective standard reaction conditions with limiting amounts of enzymes  for 5–10 min at 37°C, unless otherwise stated. For KM and kcat determination, the linear velocities were plotted against substrate concentration and the hyperbolic curve obtained fit to a rectangular hyperbola by least-squares non-linear regression method. Apparent values were obtained for the Michaelis constant, KM, and the Vmax for cleavage; kcat was calculated by dividing the Vmax by the enzyme concentration. At least three independent experiments were performed for all analyses.To measure kinetic parameters, range of duplex oligonucleotide substrate concentrations varied from 0.1 to 10 times the K−1 BSA and 1 mM DTT for the Nfo protein or the same buffer supplemented with 5 mM MgCl2 for the Apn1 protein. The activity of APE1 protein was tested either in the “BER+Mg2+” buffer containing 100 mM KCl, 20 mM HEPES-KOH (pH 7.6), 0.1 mg•mL−1 BSA, 1 mM DTT and 5 mM MgCl2 which is optimal for its AP endonuclease activity or in the “NIR buffer” containing 50 mM KCl, 20 mM HEPES-KOH (pH 6.9), 0.1 mg•mL−1 BSA, 1 mM DTT and 0.1 mM MgCl2 which is optimal for its nucleotide incision activity. When measuring APE1-NIR activity in the cell-free extracts MgCl2 was replaced with ZnCl2The standard reaction mixture (20 µL) for the NIR activity contained 50 mM KCl, 20 mM HEPES-KOH (pH 7.6), 0.1 mg•mL32P-labelled DNA substrate either in the standard DNA glycosylase BER+EDTA reaction buffer: 50 mM KCl, 20 mM HEPES-KOH (pH 7.6), 0.1 mg•mL−1 BSA, 1 mM DTT and 1 mM EDTA or in the zinc-containing “NIR+Zn2+ buffer” optimal for the APE1-catalyzed NIR activity in extracts: 50 mM KCl, 20 mM HEPES-KOH (pH 6.8), 0.1 mg•mL−1 BSA and 1 mM DTT and 0.1 mM ZnCl2E. coli cell extracts and incubated for 60 min at 37°C, unless otherwise stated.The standard incision assay in cell-free extracts (20 µl) was performed with 5 nM The reactions were stopped by adding 10 µL of a solution containing 0.5% SDS and 20 mM EDTA and then desalted by hand-made spin-down columns filled with Sephadex G25 (Amersham Biosciences) equilibrated in 7.5 M urea. Purified reaction products were heated at 65°C for 3 min and separated by electrophoresis in denaturing 20% (w/v) polyacrylamide gels . Gels were exposed to a Fuji FLA-3000 Phosphor Screen and analyzed using Image Gauge V3.12 software.In vitro reconstitution of the human NIR pathway for 5OH-Hyd residues was carried out as described previously 32P]-labelled 5OH-Hyd•G duplex was incubated in the presence of 5 nM APE1, 0.1 units POLβ, 2 nM FEN1 and 5 nM LIG1 in buffer (20 µL) containing 50 mM HEPES-KOH (pH 7.2), 30 mM NaCl, 0.1 mg/mL BSA, 2 mM DTT, 50 µM dNTPs, 2 mM ATP and 3 mM MgCl2 for 60 min at 25°C. To reveal non-repaired 5OH-Hyd residues, left after incubation with human proteins, additional treatment with E. coli Nei DNA glycosylase was performed. For this purpose, an aliquot from reaction mixture was incubated in 20 µL of DNA glycosylase buffer with 5 nM Nei for 10 min at 37°C. Reaction products were analyzed as described above.Typically, 40 pmol of lesion containing oligonucleotide duplexes (in 20 µL) were incubated with the AP endonucleases (100 ng of APE1 and/or Nfo) in the appropriate “NIR reaction buffer” (see above) at 37°C for 1 h. The products were desalted on a MicroSpin G-25 column , prior subjection to the MALDI-TOF MS measurements. The latter MALDI mass spectra in the negative mode were obtained on a time-of-flight Biflex mass spectrometer  equipped with a 337 nm nitrogen laser and pulsed delay source extraction. The matrix was prepared by dissolving 3-hydroxypicolinic acid in 10 mM ammonium citrate buffer and a small amount of Dowex-50 W 50×8–200 cation exchange resin (Sigma). Sample (1 µL) was added to matrix (1 µL) and the resulting solution was made homogeneous by stirring. The resulting sample was placed on the target plate and allowed to dry. Spectra were calibrated using reference oligonucleotides of known masses.E. coli AB1157 (leuB6 thr-1Δ(gpt-proA2) hisG4 argE3 lacY1 galK2 ara-14 mtl-1 xyl-5 thi-1 tsx-33 rpsL31 supE44 rac) (WT) and its isogenic derivatives MS2000 ) (R is the insertion of transposon Tn10 with an unknown location used as a co-transducible marker with the fpg::KnRwhere X::tet), BH130 (Rnfo::kn), BH150 (Rnth::kn), BH160 (R fpg::knRnth::kn) were from laboratory stock and SW2-8 (RΔnei::Cm) and SW2-38 (R Δnei::cmRnth::kn) isogenic to BW35 (KL16) were gifts from S. Wallace .E. coli cultures were grown to an OD600 of 1.0 in the presence or absence of 0.25 mg/mL paraquat (+P) to induce Nfo Cell-free extracts were prepared as previously described 2 in Dulbecco minimal essential medium supplemented with 10% foetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin. The EBV-transformed lymphoblastoid cell lines from wild-type (AHH1), FA-C (HSC536), FA-C expressing neomycin (HSC536N), FA-C expressing neomycin and FANCC (HSC536N/FANCC), lymphoblast cells  were routinely grown at 37°C in 5% CO2 in RPMI medium supplemented with 12% foetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin as described HeLa cells  were routinely grown at 37°C in 5% CO6 cells per Petri dish, incubated for 18 h and then transfected with the specific siRNA. Transfection efficiency was monitored by co-transfection of control cells with pmaxGFP vector  (data not shown). After 72 h, cells were collected and whole cell-free extracts were prepared as described HeLa cells were transfected with the siRNA oligonucleotides using Lipofectamine 2000  according to the manufacturer's instructions. Cells were plated at 2×10Figure S1The AP endonuclease activities towards 5OH-5Me-Hyd•A oligonucleotide duplex. The 3′-[32P]-labelled 5OH-5Me-Hyd•A oligonucleotide duplex was incubated with purified WT and mutant Nfo and APE1 under either standard condition “pH 7.6” for Nfo, or “NIR+Mg2+” same as “pH 6.9, 0.1 mM MgCl2” for APE1, or standard condition “BER+EDTA” same as “pH 7.6, 1 mM EDTA” for Fpg. (A) Denaturing PAGE analysis of the cleavage products by WT and mutant AP endonucleases. Nfo-G149G is a highly NIR-deficient mutant of Nfo; R177A and K98E are APE1 mutants with a slightly reduced NIR activity; D308A is APE1 mutant with highly reduced NIR activity; K98A/R185A is APE1 double mutant with highly reduced NIR activity; ΔN61 is a truncated APE1 mutant lacking the N-terminal 61 residues with a slightly reduced NIR activity; D210N is a catalytically inactive APE1 mutant. “5′-p-MeHyd” denotes 14-mer cleavage fragment containing 5′-terminal 5OH-5Me-Hyd residue; “5′-p-Z” denotes 14-mer cleavage fragment containing 5′-terminal ureido residue. (B) Cleavage of 5OH-5Me-Hyd•A oligonucleotide duplex by Nfo, APE1 and Fpg after 10 and 45 min incubation. “P1” and “5MeHyd-13mer” denotes 14-mer cleavage fragment containing 5′-terminal 5OH-5Me-Hyd residue; “P2” and Y-13mer” denotes 14-mer cleavage fragment containing 5′-terminal ureido residue; “P3” and “13mer” denotes 13-mer cleavage fragment containing 5′-phosphate residue. For details see (JPG)Click here for additional data file.Figure S2Comparison of the AP endonuclease activities towards urea, hydantoins and thymine glycol containing substrates. 5′-[32P]-labelled oligonucleotide duplexes were incubated with the purified WT and mutant Nfo and APE1 AP endonucleases under standard BER and NIR conditions. Nfo-G149G is a highly NIR-deficient mutant of Nfo; R177A and K98E are APE1 mutants with a slightly reduced NIR activity; D308A is APE1 mutant with highly reduced NIR activity; K98A/R185A is APE1 double mutant with highly reduced NIR activity; ΔN61 is a truncated APE1 mutant lacking the N-terminal 61 residues with a slightly reduced NIR activity; D210N is a catalytically inactive APE1 mutant. (A) Denaturing PAGE analysis of the cleavage products of Urea-RT•T, 5OH-Hyd•G and 5OH-5Me-Hyd•A oligonucleotide duplexes. Lane 1, control non-treated Urea-RT•T; lane 2, as 1 but with Nfo; lane 3, as 1 but with APE1 under NIR condition; lane 4, as 1 but with APE1 under BER condition; lane 5, as 1 but with Xth; lanes 6–10, same as 1–5 but with 5OH-Hyd•G as a substrate; lanes 11–15, same as 1–5 but with 5OH-5Me-Hyd•A as a substrate. (B) Denaturing PAGE analysis of the APE1-cleavage products of 5′-[32P]-labelled 30-mer, 34-mer and 19-mer Tg•A, oligonucleotide duplexes under NIR condition. Following reaction buffers were used for the AP endonucleases: buffer for Nfo contained 20 mM HEPES-KOH, pH 7.6, 50 mM KCl, 0.1 mg/mL BSA and 1 mM DTT treatment; buffer for APE1-NIR contained 20 mM HEPES-KOH, pH 6.9, 50 mM KCl, 0.1 mg/mL BSA, 1 mM DTT and 0.1 mM MgCl2; buffer for APE1-BER contained 20 mM HEPES-KOH, pH 7.6, 100 mM KCl, 0.1 mg/mL BSA, 1 mM DTT and 5 mM MgCl2. Reaction buffer for Xth contained 20 mM HEPES-KOH, pH 7.6, 100 mM KCl, 0.1 mg/mL BSA, 1 mM DTT and 5 mM CaCl2. Lane 1, control non-treated 30-mer Tg•A; lane 2, as 1 but with WT APE1; lane 3, as 1 but with APE1-K98E mutant; lane 4, control non-treated 34-mer Tg•A; lane 5, as 4 but with WT APE1; lane 6, as 4 but with APE1-K98E mutant; lane 7, control non-treated 19-mer 19Tg-IW•A; lane 8, as 7 but with WT APE1; lane 9, as 7 but with APE1-K98E mutant. (C) Denaturing PAGE analysis of the WT and mutant Nfo and APE1 activities on 5′-[32P]-labelled RT-THF•T and Urea-RT•T oligonucleotide duplexes. Lane 1, control non-treated RT-THF•T; lane 2, as 1 but with WT Nfo; lane 3, as 1 but with NIR-deficient mutant Nfo-G149G; lane 4, as 1 but APE1 under NIR condition; lanes 5–9, as 4 but with APE1-mutants; lanes 10–18, same as lanes 1–9 but with RT-Urea•T duplex as a substrate. For details see (JPG)Click here for additional data file.Figure S3Comparison of the AP endonucleases and DNA glycosylases activities towards pyrimidine-derived hydantoins. Enzyme concentration dependence activity curves on 5′-[32P]-labelled oligonucleotide duplexes containing hydantoins (A) Nfo, Apn1 and APE1 acting on 5OH-Hyd•G; (B) Nfo, Apn1 and APE1 acting on 5OH-5Me-Hyd•A; (C) NTH1 and NEIL1 acting on 5OH-Hyd•G; (D) NTH1 and NEIL1 acting on 5OH-5Me-Hyd•A. For details see (JPG)Click here for additional data file.Figure S4DNA repair activities towards 5OH-Hyd•G duplex in paraquat-induced E. coli cell-free extracts. 3′-[α-32P]-ddATP-labelled oligonucleotide duplexes were incubated with either 3 µg of cell-free extract or limited amount of a purified protein in the standard DNA glycosylase reaction “BER+EDTA” buffer for 30 min at 37°C. (A) Denaturing PAGE analysis of the reaction products of 5OH-Hyd•G oligonucleotide. Lanes 1 and 12, control 5OH-Hyd•G with no enzyme; lanes 2–3 and 13–14, 5OH-Hyd•G incubated with the purified proteins; lanes 4–11, 5OH-Hyd•G incubated with control non-induced extracts; lanes 15–22, 5OH-Hyd•G incubated with paraquat-induced extracts. (B) Graphic representation of the mean values of DNA repair activities on 5OH-Hyd•G. (C) Denaturing PAGE analysis of the reaction products of 5OH-5Me-Hyd•G oligonucleotide. Lanes 1 and 12, control 5OH-5Me-Hyd•A with no enzyme; lanes 2–3 and 13–14, 5OH-5Me-Hyd•A incubated with the purified proteins; lanes 4–11, 5OH-5Me-Hyd•A incubated with control non-induced extracts; lanes 15–22, 5OH-5Me-Hyd•A incubated with paraquat-induced extracts. (D) Graphic representation of the mean values of DNA repair activities on 5OH-5Me-Hyd•A. DNA glycosylase (BER) and AP endonuclease-catalyzed (NIR) incisions were calculated by measuring amount of 13-mer and 14-mer products, respectively. The background values representing control oligonucleotides degradation in absence of enzyme in lanes 1 and 12 were subtracted. “E. coli extracts+P” indicates that the expression of Nfo was induced by exposure of cell culture to 0.25 mg/mL of paraquat. For details see (JPG)Click here for additional data file.Figure S5DNA repair activities towards 5-OH-Hyd residues in the extracts of Fanconi complementation group C cells. 5′-[32P]-labelled 5OH-Hyd•G oligonucleotide duplex was incubated with 20 µg of the extracts prepared from AHH1, FA-C and FA-C+FANCC cells. (A) Denaturing PAGE analysis of the reaction products. Lane 1, 5OH-Hyd•G treated with NEIL1; lane 2, 5OH-Hyd•G treated with NTH1; lane 3, control 5OH-Hyd•G no treatment; lanes 4–6, 5OH-Hyd•G treated with AHH1 extracts; lanes 7–9, 5OH-Hyd•G treated with FA-C extracts; lanes 10–12, 5OH-Hyd•G treated with FA-C+FANCC extracts. The arrows denote the position of the 22-mer, 9-mer fragment with 3′-dRP residue (9-dRP) and 9-mer fragment with 3′-phosphate residue (9-P). (B) Graphic representation of the mean values of DNA repair activities in extracts. For details see (JPG)Click here for additional data file.


Molecular Cytogenetics had to record with the deepest regret the passing of one of its members after a long and courageous battle with cancer — Professor Anna D Polityko. She was a renowned specialist in cytogenetics, a Senior Researcher at the National Medical Center “Mother and child”, and the International Sakharov Environmental University , and the Belarus representative for the European Advisory Council of the ECA (European Cytogenetic Association). Moreover, being an active member of the Molecular Cytogenetics Editorial Board, she has significantly contributed to the recognized success of the journal.Last year, the Editorial Board of in vitro radiosensitivity in persons with balanced constitutional karyotypic abnormalities”. As a qualified specialist, Anna provided consulting services in regional genetics centers in Belarus, peformed diagnosis of chromosome-related diseases, and controlled the quality and efficiency of the latter. She contributed a lot to the fields of cytogenetics and molecular cytogenetics, being involved in numerous national and international research projects. In recognized scientific databases, more than 20 articles with her co-authorship can be found, excluding those 248 publications published in Russian.Anna D Polityko was born on December 17, 1959 in Glukhov . In 1982, she graduated from Belarusian State University  and started her career at the Institute of Hereditary Diseases of the Belarusian Ministry of Health . In 1998, she finished her PhD thesis on “Cytogenetic characteristics and On behalf of the Editorial Board, we wish to express our deepest sorrow to her family, including two children and one granddaughter, and to all her friends and colleagues. We all have lost a highly motivated colleague full of brilliant ideas, a key person in the improvement of cytogenetic/molecular cytogenetic diagnosis in her country and more importantly a very kind and supporting friend. Anna will be remembered as an exceptionally strong, enthusiastic and optimistic person. We miss her sorely.