cell-patterning-on-photolithographically-defined-parylene-c-sio2

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO<sub>2</sub> Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

The University of Edinburgh

Daily diurnal variation in admissions for ruptured abdominal aortic aneurysms.

Twenty human proteins encode Phox/Bem1p (PB1) domains, which are involved in forming protein heterodimers. MEKK2, MEKK3, and MEK5 are 3 serine-threonine protein kinases that have PB1 domains. MEKK2, MEKK3, and MEK5 are the MAP3Ks and the MAP2K in the ERK5 mitogen-activated protein kinase (MAPK) signaling module. ERK5 is a critical MAPK for both development of the vasculature and vascular homeostasis in the adult, but no other MAPK has been shown to be critical in vascular maintenance in the adult animal. MEKK2 and MEKK3 are the only MAP3Ks shown to physically interact with and activate the MEK5-ERK5 signaling module. Interaction of MEKK2 or MEKK3 with MEK5 is mediated by heterodimerization of the MEKK2 (or MEKK3) PB1 and MEK5 PB1 domains. The authors have developed a homogeneous, time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor PB1-PB1 domain heterodimerization. The assay uses a europium-chelate conjugated GST-MEK5 PB1 domain chimera, biotinylated MEKK2 PB1 domain, and streptavidin-Cy5. Interaction of the MEKK2 and MEK5 PB1 domains gives a robust FRET signal (Z' factor = 0.93), which is completely abrogated by mutation of 2 acidic residues (64D65E-->AA) within the MEK5 PB1 domain that causes loss of stable PB1-PB1 domain interaction. This assay can be used to study the specificity of PB1-PB1 domain interactions and to screen for molecules that can regulate MEKK2/MEKK3-MEK5 interactions. Disruption of PB1 domain interactions represents a novel approach for selectively regulating the ERK5 signaling pathway independent of kinase active site-directed adenosine triphosphate competitive inhibitors.

Homogeneous time-resolved fluorescence resonance energy transfer assay for measurement of Phox/Bem1p (PB1) domain heterodimerization.

The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectious diseases. Toward the goal of validating RecA as a novel target in the chemotherapy of bacterial infections, the authors have screened 35,780 small molecules against RecA. In total, 80 small molecules were identified as primary hits and could be clustered in 6 distinct chemotype clades. The most potent class of hits was further examined, and 1 member compound was found to inhibit RecA-mediated strand exchange and prevent ciprofloxacin-induced SOS expression in Escherichia coli. This compound represents the first small molecule demonstrating an ability to inhibit the bacterial SOS response in live bacterial cell cultures.

Inhibitors of RecA activity discovered by high-throughput screening: cell-permeable small molecules attenuate the SOS response in Escherichia coli.

We report emission from a bismuth doped chalcogenide glass which is flattened, has a full width at half maximum (FWHM) of 600 nm, peaks at 1300 nm and covers the entire telecommunications window. At cryogenic temperatures the FWHM reaches 850 nm. The quantum efficiency and lifetime were as high as 32% and 175 mus, respectively. We also report two new bismuth emission bands at 2000 and 2600 nm. Absorption bands at 680, 850, 1020 and 1180 nm were observed. The 1180 nm absorption band was previously unobserved. We suggest that the origin of the emission in Bi:GLS is Bi22- dimers.

Ultrabroad emission from a bismuth doped chalcogenide glass.

The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents.

Novel Inhibitors of E. coli RecA ATPase Activity.

Seasonal variation in incidence of spontaneous subarachnoid haemorrhage (SAH) is well recognised. This retrospective single-centre study aimed to characterise seasonality of SAH in the temperate climate of London, UK and to determine associations of incidence with meteorological variables. We further investigated whether associations vary according to location of aneurysm.

A 5-year retrospective study assessing the association between seasonal and meteorological change and incidence of aneurysmal subarachnoid haemorrhage.

Our professional and personal lives depend increasingly on access to information via the Internet. As an open access resource, the Internet is on the whole unbridled by censorship and can facilitate the rapid propagation of ideas and discoveries. At the same time, this liberty in sharing information, being unregulated and often free from external validation, can be oppressive; overloading the user and hindering effective decision-making. It can be difficult, if not impossible, to reliably ascertain the provenance of data and opinion. We must, therefore, discern what is useful, relevant, and above all reliable if we are to harness the Internet's potential to improve training, delivery of care, research, and provision of patient information. This article profiles the resources currently available to neurosurgeons, asks how we can sort the informational wheat from the chaff, and explores where future developments might further influence neurosurgical practice.

The Internet for neurosurgeons: current resources and future challenges.

CPT-11 is a widely-used anti-cancer drug that is converted in vivo to its active metabolite, SN-38. In the liver, enzymes detoxify SN-38 by coupling it to a glucuronidate moiety and this inactive compound (SN-38G) is excreted into the gastrointestinal tract. In the intestine, commensal bacteria convert the SN-38G back to the active and toxic SN-38 using bacterial β-glucuronidase enzyme (GUS). This intestinal SN-38 causes debilitating diarrhea that prevents dose-intensification and efficacy in a significant fraction of patients undergoing CPT-11 treatment for cancer. This CPT-11 metabolic pathway suggests that small molecule inhibitors of GUS may have utility as novel therapeutics for prevention of dose-limiting diarrhea resulting from CPT-11 therapy. To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library. Novel inhibitors of GUS were identified with IC(50) values ranging from 50 nM to 4.8 µM. These compounds may be useful as chemical probes for use in proof-of-concept experiments designed to determine the efficacy of GUS inhibitors in altering the intestinal metabolism of drugs. Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.

A High Throughput Assay for Discovery of Bacterial β-Glucuronidase Inhibitors.

Confident intra-operative localisation of thoracic spinal pathology remains challenging. Several strategies are routinely employed, including intra-operative fluoroscopy and pre-operative image-guided skin marking. These techniques are limited both by potential inaccuracy and inconvenience. Here we present a novel, efficient and accurate technique for intra-operative localisation of thoracic spinal pathology using pre-operative CT-guided placement of a flexible hook-wire marker.

Intra-operative localisation of spinal level using pre-operative CT-guided placement of a flexible hook-wire marker.

The active metabolite of the chemotherapeutic irinotecan, SN-38, is detoxified through glucuronidation and then excreted into the gastrointestinal tract. Intestinal bacteria convert the glucuronidated metabolite back to the toxic SN-38 using β-glucuronidase (GUS), resulting in debilitating diarrhea. Inhibiting GUS activity may relieve this side effect of irinotecan. In this study, we sought to determine whether any known drugs have GUS inhibitory activity. We screened a library of Food and Drug Administration-approved drugs with a cell-free biochemical enzyme assay using purified bacterial GUS. After triage, five drugs were confirmed to inhibit purified bacterial GUS. Three of these were the monoamine oxidase inhibitors nialamide, isocarboxazid, and phenelzine with average IC(50) values for inhibiting GUS of 71, 128, and 2300 nM, respectively. The tricyclic antidepressant amoxapine (IC(50) = 388 nM) and the antimalarial mefloquine (IC(50) = 1.2 µM) also had activity. Nialamide, isocarboxazid, and amoxapine had no significant activity against purified mammalian GUS but showed potent activity for inhibiting endogenous GUS activity in a cell-based assay using living intact Escherichia coli with average IC(50) values of 17, 336, and 119 nM, respectively. Thus, nialamide, isocarboxazid, and amoxapine have potential to be repurposed as therapeutics to reduce diarrhea associated with irinotecan chemotherapy and warrant further investigation for this use.

Potential repurposing of known drugs as potent bacterial β-glucuronidase inhibitors.

MEKK2 &#x6FC0;&#x9176; (MAP3K2) &#x6700;&#x8FD1;&#x4E00;&#x76F4;&#x5728;&#x53C2;&#x4E0E;&#x80BF;&#x7624;&#x751F;&#x957F;&#x548C;&#x8F6C;&#x79FB;&#x3002;&#x56E0;&#x6B64;&#xFF0C;MEKK2 &#x7684;&#x9009;&#x62E9;&#x6027;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x53EF;&#x80FD;&#x662F;&#x80BF;&#x7624;&#x6CBB;&#x7597;&#x7684;&#x65B0;&#x7B56;&#x7565;&#x3002;&#x82E5;&#x8981;&#x6807;&#x8BC6; MEKK2 &#x6FC0;&#x9176;&#x6D3B;&#x6027;&#x7684;&#x6291;&#x5236;&#x5242;&#xFF0C;&#x6211;&#x4EEC;&#x5F00;&#x53D1;&#x4E86;&#x65B0;&#x578B;&#x6D3B;&#x6027;&#x6D4B;&#x5B9A;&#x4E3A; MEKK2 &#x57FA;&#x4E8E;&#x91CD;&#x7EC4;&#x7EAF;&#x51C0;&#x7684; MEKK2 &#x5177;&#x6709;&#x5185;&#x5728; ATPase &#x6D3B;&#x6027;&#x7684;&#x53D1;&#x73B0;&#x3002;&#x8FD9;&#x79CD; MEKK2 ATPase &#x6D4B;&#x5B9A;&#x65B9;&#x6CD5;&#x9A8C;&#x8BC1;&#x5176;&#x9176;&#x6807;&#x8BC6;&#x548C;&#x9176;&#x7EAF;&#x5EA6;&#x7531;&#x591A;&#x4E2A;&#x65B9;&#x6CD5;&#x5305;&#x62EC;&#x8D28;&#x8C31;&#x5206;&#x6790;&#x3001; &#x6D4B;&#x8BD5;&#x4E0D;&#x540C;&#x6765;&#x6E90;&#x7684; MEKK2 &#x548C; ATPase &#x6D4B;&#x5B9A; IC(50) &#x6570;&#x636E;&#x591A;&#x6291;&#x5236;&#x5242;&#x5BF9;&#x6587;&#x5B66;&#x4EF7;&#x503C;&#x548C;&#x4F7F;&#x7528; MEKK2 &#x7ED1;&#x5B9A;&#x548C; transphosphorylation &#x68C0;&#x5B9A;&#x6CD5;&#x751F;&#x6210;&#x7684; IC(50) &#x6570;&#x636E;&#x8FDB;&#x884C;&#x6BD4;&#x8F83;&#x3002;&#x4E24;&#x8005;&#x5408;&#x8BA1;&#xFF0C;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E;&#x771F;&#x6B63;&#x7684; MEKK2 &#x6D3B;&#x52A8;&#x6B63;&#x5728;&#x8861;&#x91CF;&#x8FD9;&#x79CD;&#x6D4B;&#x5B9A;&#x65B9;&#x6CD5;&#x4E2D;&#xFF0C;&#x5E76;&#x6CA1;&#x6709;&#x5176;&#x4ED6;&#x4E09;&#x6709;&#x52A9;&#x4E8E;&#x4FE1;&#x53F7;&#x3002;&#x6D4B;&#x5B9A;&#x7684;&#x5FAE;&#x578B;&#x7684;&#x7248;&#x672C;&#xFF0C;&#x9AD8;&#x901A;&#x91CF;&#x7B5B;&#x9009;&#x3001; &#x9274;&#x5B9A;&#x548C;&#x590D;&#x5408;&#x56FE;&#x4E66;&#x9986;&#x88AB;&#x5C4F;&#x853D;&#x3002;&#x7B5B;&#x9009;&#x4E2D;&#x7684;&#x70B9;&#x51FB;&#x7387;&#x5728; MEKK2 &#x5185;&#x5728; ATPase&#x3001; &#x7ED1;&#x5B9A;&#x548C; transphosphorylation &#x5316;&#x9A8C;&#x4E2D;&#x751F;&#x6210;&#x7C7B;&#x4F3C;&#x6548;&#x529B;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x4E00;&#x79CD;&#x65B0;&#x578B;&#x7684; MEKK2 &#x6291;&#x5236;&#x5242;&#xFF0C;&#x5E76;&#x8BC1;&#x5B9E; crizotinib &#x548C; bosutinib &#x4E0E; IC(50) &#x7684;&#x503C;&#xFF0C;&#x70C8;&#x6027;&#x4F53;&#x5916;&#x6291;&#x5236;&#x5242; MEKK2 &#x6D3B;&#x52A8; &lt; 100 nM&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x8FD9;&#x79CD;&#x6D4B;&#x5B9A;&#x65B9;&#x6CD5;&#x6709;&#x7684;&#x5C0F;&#x5206;&#x5B50;&#x6291;&#x5236;&#x5242; MEKK2 &#x6D3B;&#x52A8;&#x53D1;&#x73B0;&#x7684;&#x5B9E;&#x7528;&#x7A0B;&#x5E8F;&#x3002;

&#x53D1;&#x5C55;&#x548C;&#x9AD8;&#x541E;&#x5410;&#x91CF;&#x5185;&#x5728; ATPase &#x6D3B;&#x6027;&#x6D4B;&#x5B9A;&#x4E3A; MEKK2 &#x6291;&#x5236;&#x5242;&#x7684;&#x53D1;&#x73B0;&#x9A8C;&#x8BC1;&#x3002;

The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. To identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC(50) data for multiple inhibitors to literature values and to IC(50) data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high-throughput screening, and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding, and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC(50) values of <100 nM. Thus, this assay has utility for the discovery of small-molecule inhibitors of MEKK2 activity.

Development and Validation of a High-Throughput Intrinsic ATPase Activity Assay for the Discovery of MEKK2 Inhibitors.

La kinase MEKK2 (MAP3K2) a r&eacute;cemment &eacute;t&eacute; impliqu&eacute; dans la croissance tumorale et la m&eacute;tastase. Ainsi, l&#39;inhibition s&eacute;lective de la MEKK2 peut &ecirc;tre une strat&eacute;gie novatrice pour le traitement du cancer. Pour identifier les inhibiteurs de l&#39;activit&eacute; de la kinase MEKK2, nous avons d&eacute;velopp&eacute; un test d&#39;activit&eacute; nouvelle pour MEKK2 bas&eacute;e sur la d&eacute;couverte que MEKK2 purifi&eacute; recombinant a l&#39;activit&eacute; ATPasique intrins&egrave;que. Ce dosage MEKK2 ATPase a &eacute;t&eacute; valid&eacute; pour l&#39;identit&eacute; de l&#39;enzyme et la puret&eacute; enzymatique par plusieurs m&eacute;thodes comprenant l&#39;analyse de spectrom&eacute;trie de masse, les essais de diff&eacute;rentes sources de MEKK2 et comparant ATPase test IC (50) donn&eacute;es pour plusieurs inhibiteurs aux valeurs de la litt&eacute;rature et aux donn&eacute;es d&#39;IC (50) g&eacute;n&eacute;r&eacute;es &agrave; l&#39;aide de la liaison MEKK2 et transphosphorylation des essais. Pris ensemble, ces donn&eacute;es indiquent que v&eacute;ritable activit&eacute; MEKK2 a &eacute;t&eacute; mesur&eacute;e dans ce test et aucun autres ATPases ne contribua au signal. Une version miniaturis&eacute;e du test a &eacute;t&eacute; valid&eacute;e pour high-throughput screening et banques de compos&eacute;s ont &eacute;t&eacute; examin&eacute;s. Les visites de d&eacute;pistage g&eacute;n&eacute;r&eacute; des puissances comparables dans le MEKK2 ATPasique intrins&egrave;que, liaison et des essais de transphosphorylation. Nous avons identifi&eacute; un nouvel inhibiteur de la MEKK2 et a confirm&eacute; que crizotinib et bosutinib sont de puissants inhibiteurs in vitro de l&#39;activit&eacute; de la MEKK2 avec IC (50) les valeurs de &lt; 100 nM. Ainsi, ce test a une utilit&eacute; pour la d&eacute;couverte de petites mol&eacute;cules inhibitrices de l&#39;activit&eacute; MEKK2.

D&eacute;veloppement et Validation d&#39;un dosage de l&#39;activit&eacute; ATPasique intrins&egrave;que de haut d&eacute;bit pour la d&eacute;couverte des inhibiteurs de la MEKK2.

Die Kinase MEKK2 (MAP3K2) hat vor kurzem im Tumorwachstum und Metastasierung impliziert worden. Selektive Hemmung der MEKK2 kann so eine neuartige Strategie f&uuml;r Krebstherapie sein. Um Inhibitoren der MEKK2-Kinase-Aktivit&auml;t zu identifizieren, entwickelten wir eine neuartige Aktivit&auml;tstest f&uuml;r MEKK2 basierend auf der Entdeckung, dass rekombinante gereinigten MEKK2 systeminterne ATPase-Aktivit&auml;t hat. Diese MEKK2 ATPase-Assay wurde durch mehrere Methoden einschlie&szlig;lich der Massenspektrometrie-Analyse, Tests von verschiedene Quellen von MEKK2 und Vergleich der ATPase Assay IC (50) Daten f&uuml;r mehrere Hemmer Literatur-Werte und IC (50) Daten mit MEKK2 Bindung und Transphosphorylation Assays generiert f&uuml;r Enzym-Identit&auml;t und enzymatische Reinheit &uuml;berpr&uuml;ft. Zusammengenommen ergab diese Daten, dass echte MEKK2-Aktivit&auml;t in dieser Probe gemessen wurde und keine anderen ATPasen dazu, das Signal beigetragen. Eine miniaturisierte Version des Tests wurden f&uuml;r Hochdurchsatz-screening abgeglichen, und zusammengesetzte Bibliotheken wurden gezeigt. Die Screening-Hits generiert vergleichbare Potenzen in der MEKK2 systeminterne ATPase, Bindung und Transphosphorylation Proben. Wir identifiziert eine neuartige MEKK2-Inhibitor und best&auml;tigt, Crizotinib und Bosutinib potente in-vitro-Inhibitoren der MEKK2 Aktivit&auml;t mit IC (50) Werte von &lt; 100 nM. So hat diese Probe Dienstprogramm f&uuml;r die Entdeckung von klein-Molek&uuml;l-Inhibitoren der MEKK2 T&auml;tigkeit.

Entwicklung und Validierung einer Hochdurchsatz systeminterne ATPase-Aktivit&auml;tstest zur Entdeckung des MEKK2-Inhibitoren.

La chinasi MEKK2 (MAP3K2) recentemente &egrave; stato implicato nella crescita tumorale e le metastasi. Cos&igrave;, l&#39;inibizione selettiva della MEKK2 pu&ograve; essere una romanzo strategia per la terapia del cancro. Per identificare inibitori dell&#39;attivit&agrave; della chinasi MEKK2, abbiamo sviluppato un saggio romanzo attivit&agrave; per MEKK2 basato sulla scoperta che MEKK2 ricombinante purificata ha intrinseca attivit&agrave; ATPasi. Questo dosaggio MEKK2 ATPasi &egrave; stato convalidato per l&#39;enzima identit&agrave; e purezza enzimatica da pi&ugrave; metodi, tra cui analisi di spettrometria di massa, prova diverse fonti di MEKK2 e confrontando ATPasi dosaggio IC(50) dati per pi&ugrave; inibitori di valori di letteratura e di IC(50) dati generati utilizzando MEKK2 associazione e dosaggi di transphosphorylation. Presi insieme, questi dati indicato che genuino MEKK2 attivit&agrave; era essere misurata in questo dosaggio e non altre ATPasi ha contribuito al segnale. Una versione miniaturizzata del dosaggio &egrave; stata convalidata per high-throughput screening, e librerie di composte sono state proiettate. The hits screening generato potenze comparabili nel MEKK2 intrinseca ATPasi, associazione e dosaggi di transphosphorylation. Abbiamo identificato un inibitore del romanzo MEKK2 e ha confermato che il crizotinib e bosutinib sono potenti inibitori in vitro dell&#39;attivit&agrave; MEKK2 con valori IC(50) di &lt; 100 nM. Quindi, questo dosaggio ha una utilit&agrave; per la scoperta della piccolo-molecola inibitori dell&#39;attivit&agrave; MEKK2.

Sviluppo e validazione di un test di attivit&agrave; ATPasi intrinseca High-Throughput per la scoperta degli inibitori di MEKK2.

&#x30AD;&#x30CA;&#x30FC;&#x30BC; MEKK2 (MAP3K2) &#x304C;&#x6700;&#x8FD1;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x816B;&#x760D;&#x306E;&#x5897;&#x6B96;&#x3068;&#x8EE2;&#x79FB;&#x3067;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;MEKK2 &#x306E;&#x9078;&#x629E;&#x7684;&#x963B;&#x5BB3;&#x764C;&#x7642;&#x6CD5;&#x306E;&#x305F;&#x3081;&#x306E;&#x65B0;&#x6226;&#x7565;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;MEKK2 &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x306E;&#x6291;&#x5236;&#x5264;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x6211;&#x3005; &#x306E;&#x7D44;&#x63DB;&#x3048;&#x306E;&#x6D44;&#x5316;&#x3055;&#x308C;&#x305F; MEKK2 &#x56FA;&#x6709;&#x306E; ATPase &#x6D3B;&#x6027;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x767A;&#x898B;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066; MEKK2 &#x65B0;&#x898F;&#x6D3B;&#x6027;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3092;&#x958B;&#x767A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E; MEKK2 &#x306E; atpase &#x6D3B;&#x6027;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x9175;&#x7D20;&#x30A2;&#x30A4;&#x30C7;&#x30F3;&#x30C6;&#x30A3;&#x30C6;&#x30A3;&#x3068;&#x9175;&#x7D20;&#x306E;&#x7D14;&#x5EA6;&#x306E;&#x8CEA;&#x91CF;&#x5206;&#x6790;&#x3092;&#x542B;&#x3080;&#x3001;&#x7570;&#x306A;&#x308B;&#x30BD;&#x30FC;&#x30B9; MEKK2 &#x306E;&#x30C6;&#x30B9;&#x30C8;&#x304A;&#x3088;&#x3073; atpase &#x6D3B;&#x6027;&#x30A2;&#x30C3;&#x30BB;&#x30A4; ic50 &#x5024; &#xFF08;50 &#x30C7;&#x30FC;&#x30BF;&#x8907;&#x6570;&#x963B;&#x5BB3;&#x5264;&#x6587;&#x732E;&#x5024;&#x3068; MEKK2 &#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x3068;&#x308A;&#x3093;&#x9178;&#x8EE2;&#x79FB;&#x53CD;&#x5FDC;&#x306E;&#x8A66;&#x91D1;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F; ic50 &#x5024; &#xFF08;50 &#x30C7;&#x30FC;&#x30BF;&#x3092;&#x6BD4;&#x8F03;&#x3059;&#x308B;&#x8907;&#x6570;&#x306E;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x3067;&#x691C;&#x8A3C;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x4E00;&#x7DD2;&#x306B;&#x53D6;&#x3089;&#x308C;&#x3066;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x672C;&#x7269;&#x306E; MEKK2 &#x6D3B;&#x52D5;&#x306F;&#x3001;&#x3053;&#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3067;&#x6E2C;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x305F;&#x3057;&#x3001;&#x4FE1;&#x53F7;&#x3092;&#x4ED6; Atpase &#x8CA2;&#x732E;&#x3057;&#x3066;&#x3044;&#x306A;&#x3044;&#x793A;&#x3057;&#x305F;&#x3002;&#x5C0F;&#x578B;&#x7248;&#x3001;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8; &#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3001;&#x305F;&#x3081;&#x691C;&#x8A3C;&#x3055;&#x308C;&#x5316;&#x5408;&#x7269;&#x30E9;&#x30A4;&#x30D6;&#x30E9;&#x30EA;&#x304C;&#x4E0A;&#x6620;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x5B89;&#x6253;&#x5339;&#x6575;&#x30DD;&#x30FC;&#x30C6;&#x30F3;&#x30B7;&#x30FC; MEKK2 &#x7D44;&#x307F;&#x8FBC;&#x307F; ATPase &#x30B5;&#x30FC;&#x30D3;&#x30B9;&#x3001;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3001;&#x304A;&#x3088;&#x3073;&#x308A;&#x3093;&#x9178;&#x8EE2;&#x79FB;&#x53CD;&#x5FDC;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3092;&#x751F;&#x6210;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x65B0;&#x898F; MEKK2 &#x963B;&#x5BB3;&#x5264;&#x3092;&#x8B58;&#x5225;&#x3057;&#x3001;&#x30AF;&#x30EA;&#x30BE;&#x30C1;&#x30CB;&#x30D6;&#x3068; bosutinib &#x306E; ic50 &#x5024; &#xFF08;50 &#x5024;&#x3092;&#x6301;&#x3064; MEKK2 &#x6D3B;&#x52D5;&#x306E;&#x5F37;&#x529B;&#x306A; in vitro &#x963B;&#x5BB3;&#x5264;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x305F; &lt; 100 nM&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x3053;&#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x5C0F;&#x5206;&#x5B50;&#x963B;&#x5BB3;&#x5264; MEKK2 &#x306E;&#x6D3B;&#x52D5;&#x306E;&#x767A;&#x898B;&#x306E;&#x305F;&#x3081;&#x306E;&#x30E6;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30C6;&#x30A3;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x958B;&#x767A;&#x3068;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8;&#x306E;&#x672C;&#x8CEA;&#x7684; atp &#x30A2;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E; MEKK2 &#x963B;&#x5BB3;&#x5264;&#x306E;&#x767A;&#x898B;&#x306E;&#x691C;&#x8A3C;&#x3002;

&#xD0A4; MEKK2 (MAP3K2) &#xCD5C;&#xADFC;&#xC5D0; &#xC885;&#xC591; &#xC131;&#xC7A5; &#xBC0F; &#xC804;&#xC774;&#xC5D0; &#xC5F0;&#xB8E8; &#xB418;&#xC5B4;&#xC788;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, mekk2&#xC758; &#xC120;&#xD0DD;&#xC801; &#xC800;&#xD574; &#xC554; &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xC804;&#xB7B5;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. MEKK2 &#xD0A4; &#xB2C8; &#xC544; &#xC81C; &#xD65C;&#xB3D9;&#xC758; &#xC800; &#xD574;&#xC81C;&#xB97C; &#xC2DD;&#xBCC4; &#xD558;&#xB824;&#xBA74; MEKK2 &#xC7AC;&#xC870;&#xD569; &#xC21C;&#xD654; mekk2&#xB294; &#xB0B4;&#xC7A5; ATPase &#xD65C;&#xB3D9; &#xBC1C;&#xACAC;&#xC5D0; &#xAE30;&#xBC18;&#xC5D0; &#xB300; &#xD55C; &#xC18C;&#xC124; &#xD65C;&#xB3D9; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xAC1C;&#xBC1C; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; MEKK2 ATPase &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xC9C8;&#xB7C9; &#xBD84;&#xC11D; &#xBD84;&#xC11D;&#xC744; &#xD3EC;&#xD568; &#xD55C; mekk2&#xC758; &#xB2E4;&#xB978; &#xC18C;&#xC2A4;&#xB97C; &#xD14C;&#xC2A4;&#xD2B8; &#xD558; &#xACE0; ATPase &#xC5EC;&#xB7EC; &#xC5B5;&#xC81C; &#xBB38;&#xD559; &#xAC12;&#xACFC; MEKK2 &#xBC14;&#xC778;&#xB529; &#xBC0F; transphosphorylation &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC0DD;&#xC131; &#xB41C; IC(50) &#xB370;&#xC774;&#xD130;&#xC5D0; &#xB300; &#xD55C; &#xBD84;&#xC11D; &#xACB0;&#xACFC; IC(50) &#xB370;&#xC774;&#xD130; &#xBE44;&#xAD50; &#xD558;&#xB294; &#xC5EC;&#xB7EC; &#xBC29;&#xBC95;&#xC73C;&#xB85C; &#xD6A8;&#xC18C; &#xC815;&#xCCB4;&#xC131;&#xACFC; &#xD6A8;&#xC18C; &#xC21C;&#xB3C4; &#xB300; &#xD55C; &#xC720;&#xD6A8;&#xC131;&#xC744; &#xAC80;&#xC0AC; &#xD588;&#xB2E4;. &#xD568;&#xAED8; &#xCC0D;&#xC740;, &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; &#xD45C;&#xC2DC; &#xC815;&#xD488; MEKK2 &#xD65C;&#xB3D9;&#xC740;&#xC774; &#xBD84;&#xC11D; &#xACB0;&#xACFC;&#xC5D0;&#xC11C; &#xCE21;&#xC815; &#xB418;&#xB294; &#xBC0F; &#xB2E4;&#xB978; ATPases &#xC2E0;&#xD638;&#xC5D0; &#xAE30;&#xC5EC;. &#xBD84;&#xC11D; &#xACB0;&#xACFC;&#xC758; &#xC18C;&#xD615;&#xB41C; &#xBC84;&#xC804; &#xC0C1;&#xC601;, &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9;&#xC5D0; &#xB300; &#xD55C; &#xC720;&#xD6A8;&#xC131;&#xC744; &#xAC80;&#xC0AC; &#xD558; &#xACE0; &#xD654;&#xD569;&#xBB3C; &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC; &#xC0C1;&#xC601; &#xD588;&#xB2E4;. &#xC2EC;&#xC0AC; &#xC548;&#xD0C0; MEKK2 &#xB0B4;&#xC7A5; ATPase, &#xBC14;&#xC778;&#xB529;, &#xBC0F; transphosphorylation &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC5D0; &#xBE44;&#xD574; potencies &#xC0DD;&#xC131;. &#xC6B0;&#xB9AC; &#xC18C;&#xC124; MEKK2 &#xC5B5;&#xC81C;&#xC81C;&#xB97C; &#xC2DD;&#xBCC4; &#xD558; &#xACE0; crizotinib&#xC640; bosutinib&#xC758; IC(50) &#xAC12;&#xACFC; MEKK2 &#xD65C;&#xB3D9;&#xC758; &#xAC15;&#xB825;&#xD55C; &#xC0DD;&#xCCB4; &#xC678;&#xC5D0;&#xC11C; &#xC800; &#xD574;&#xC81C;&#xB294; &#xD655;&#xC778; &lt; 100 nM. &#xB530;&#xB77C;&#xC11C;,&#xC774; &#xBD84;&#xC11D; &#xACB0;&#xACFC; MEKK2 &#xD65C;&#xB3D9;&#xC758; &#xC791;&#xC740; &#xBD84;&#xC790; &#xC5B5;&#xC81C;&#xC81C;&#xC758; &#xBC1C;&#xACAC;&#xC5D0; &#xB300; &#xD55C; &#xC720;&#xD2F8;&#xB9AC;&#xD2F0;.

&#xAC1C;&#xBC1C; &#xBC0F; MEKK2 &#xC800; &#xD574;&#xC81C;&#xC758; &#xBC1C;&#xACAC;&#xC5D0; &#xB300; &#xD55C; &#xB192;&#xC740; &#xC2A4;&#xB8E8;&#xD48B; &#xB0B4;&#xC7A5; ATPase &#xD65C;&#xB3D9; &#xBD84;&#xC11D; &#xACB0;&#xACFC;&#xC758; &#xC720;&#xD6A8;&#xC131;&#xC744; &#xAC80;&#xC0AC; &#xD569;&#xB2C8;&#xB2E4;.

La quinasa MEKK2 (MAP3K2) recientemente se ha implicado en el crecimiento tumoral y la met&aacute;stasis. As&iacute;, la inhibici&oacute;n selectiva de la MEKK2 puede ser una nueva estrategia para el tratamiento del c&aacute;ncer. Para identificar inhibidores de quinasa MEKK2, hemos desarrollado un ensayo de actividad novedosa para MEKK2 basadas en el descubrimiento que recombinante MEKK2 purificada tiene actividad intr&iacute;nseca de la ATPasa. Este ensayo de MEKK2 ATPasa se valid&oacute; la enzima identidad y pureza enzim&aacute;tica por varios m&eacute;todos, incluyendo an&aacute;lisis de espectrometr&iacute;a de masas, prueba diferentes fuentes de MEKK2 y comparaci&oacute;n de los datos IC(50) de ensayo ATPasa para inhibidores m&uacute;ltiples valores de la literatura y datos IC(50) mediante el enlace MEKK2 y ensayos de transfosforilaci&oacute;n. Tomados en conjunto, estos datos indicaron que genuino MEKK2 actividad se midi&oacute; en este ensayo y no otras ATPasas contribuyeron a la se&ntilde;al. Una versi&oacute;n miniaturizada del ensayo se valid&oacute; para detecci&oacute;n de alto rendimiento, y se revisaron las bibliotecas compuestas. Los &eacute;xitos de proyecci&oacute;n generan potencias comparables en la MEKK2 intr&iacute;nseca ATPasa, vinculante y ensayos transfosforilaci&oacute;n. Se identificaron un nuevo inhibidor de la MEKK2 y confirm&oacute; que potentes inhibidores in vitro de actividad MEKK2 con valores IC(50) de crizotinib y bosutinib &lt; 100 nM. As&iacute;, este ensayo tiene utilidad para el descubrimiento de mol&eacute;culas peque&ntilde;as de inhibidores de la actividad MEKK2.

Desarrollo y validaci&oacute;n de un an&aacute;lisis de actividad de la ATPasa intr&iacute;nseca de alto rendimiento para el descubrimiento de los inhibidores de la MEKK2.

Pre-operative warm up: are we missing an opportunity to improve performance? A questionnaire.

Reaction order in Bi-doped oxide glasses depends on the optical basicity of the glass host. Red and NIR photoluminescence (PL) bands result from Bi(2+) and Bin clusters, respectively. Very similar centers are present in Bi- and Pb-doped oxide and chalcogenide glasses. Bi-implanted and Bi melt-doped chalcogenide glasses display new PL bands, indicating that new Bi centers are formed. Bi-related PL bands have been observed in glasses with very similar compositions to those in which carrier-type reversal has been observed, indicating that these phenomena are related to the same Bi centers, which we suggest are interstitial Bi(2+) and Bi clusters.

On the analogy between photoluminescence and carrier-type reversal in Bi- and Pb-doped glasses.

Interfacing neurons with silicon semiconductors is a challenge being tackled through various bioengineering approaches. Such constructs inform our understanding of neuronal coding and learning and ultimately guide us toward creating intelligent neuroprostheses. A fundamental prerequisite is to dictate the spatial organization of neuronal cells. We sought to pattern neurons using photolithographically defined arrays of polymer parylene-C, activated with fetal calf serum. We used a purified human neuronal cell line [Lund human mesencephalic (LUHMES)] to establish whether neurons remain viable when isolated on-chip or whether they require a supporting cell substrate. When cultured in isolation, LUHMES neurons failed to pattern and did not show any morphological signs of differentiation. We therefore sought a cell type with which to prepattern parylene regions, hypothesizing that this cellular template would enable secondary neuronal adhesion and network formation. From a range of cell lines tested, human embryonal kidney (HEK) 293 cells patterned with highest accuracy. LUHMES neurons adhered to pre-established HEK 293 cell clusters and this coculture environment promoted morphological differentiation of neurons. Neurites extended between islands of adherent cell somata, creating an orthogonally arranged neuronal network. HEK 293 cells appear to fulfill a role analogous to glia, dictating cell adhesion, and generating an environment conducive to neuronal survival. We next replaced HEK 293 cells with slower growing glioma-derived precursors. These primary human cells patterned accurately on parylene and provided a similarly effective scaffold for neuronal adhesion. These findings advance the use of this microfabrication-compatible platform for neuronal patterning. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Article	Year
Daily diurnal variation in admissions for ruptured abdominal aortic aneurysms. World journal of surgery\| PubMed ID: 18064511	2008
Homogeneous time-resolved fluorescence resonance energy transfer assay for measurement of Phox/Bem1p (PB1) domain heterodimerization. Journal of biomolecular screening\| PubMed ID: 18480472	2008
Inhibitors of RecA activity discovered by high-throughput screening: cell-permeable small molecules attenuate the SOS response in Escherichia coli. Journal of biomolecular screening\| PubMed ID: 19675313	2009
Ultrabroad emission from a bismuth doped chalcogenide glass. Optics express\| PubMed ID: 19997155	2009
Novel Inhibitors of E. coli RecA ATPase Activity. Current chemical genomics\| PubMed ID: 20648224	2010
A 5-year retrospective study assessing the association between seasonal and meteorological change and incidence of aneurysmal subarachnoid haemorrhage. British journal of neurosurgery\| PubMed ID: 20726749	2010
The Internet for neurosurgeons: current resources and future challenges. British journal of neurosurgery\| PubMed ID: 21501057	2011
A High Throughput Assay for Discovery of Bacterial β-Glucuronidase Inhibitors. Current chemical genomics\| PubMed ID: 21643506	2011
Intra-operative localisation of spinal level using pre-operative CT-guided placement of a flexible hook-wire marker. British journal of neurosurgery\| PubMed ID: 21707263	2011
Potential repurposing of known drugs as potent bacterial β-glucuronidase inhibitors. Journal of biomolecular screening\| PubMed ID: 22535688	2012
Development and Validation of a High-Throughput Intrinsic ATPase Activity Assay for the Discovery of MEKK2 Inhibitors. Journal of biomolecular screening\| PubMed ID: 23134735	2012
Pre-operative warm up: are we missing an opportunity to improve performance? A questionnaire. International journal of surgery (London, England)\| PubMed ID: 23499903	2013
On the analogy between photoluminescence and carrier-type reversal in Bi- and Pb-doped glasses. Optics express\| PubMed ID: 23571900	2013
Patterning human neuronal networks on photolithographically engineered silicon dioxide substrates functionalized with glial analogues. Journal of biomedical materials research. Part A\| PubMed ID: 23733444	2013

Mark A. Hughes

Biography

JoVE Journal Publications

Other Publications