Combinatorial Anti-arenaviral Therapy with the Small Molecule SKI-1/S1P Inhibitor PF-429242 and Ribavirin

Arenaviruses are enveloped negative strand viruses that cause acute and chronic infections. Several Arenaviruses can cause severe hemorrhagic fever in humans. In West Africa Lassa virus causes several hundred thousand infections per year, while Junin, Machupo, Guanarito, and Sabia virus have emerged in South America. So far, only one drug is licensed against arenaviruses, the nucleoside analogue Ribavirin (Rib), which is effective when given early in disease, but shows only minor therapeutic effects in late stages of the infection. Previous works demonstrated that processing of the arenavirus glycoprotein precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexinisozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infectionand production of infectious virus. Recently, the SKI-1/S1P inhibitor PF-429242wasshownto inhibit Old World arenavirusGPCprocessing, cell-to-cell propagation, and infectious virus production. In the present study, we assessed the activity of PF-429242 against processing of the GPCs of the genetically and structurally more distant New World arenaviruses and found potent inhibition of processing of the GPCs of Junin, Machupo, and Guanarito virus. Using the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we studied the potency of PF-429242 in the context of acute and chronic infection. In line with published data, PF-429242 potently inhibited acute LCMV infection. PF-429242 was also highly active against chronic infection and drug treatment resulted in rapid extinction of the virus without emergence of drug-resistant variants. In a combinatorial drug approach, we found that PF-429242 potentiated the anti-viral effect of Rib in treatment of acute andchronic infection. Taken together, we showed that the SKI-1/S1P inhibitor PF-429242 is broadly active against GPC processing of all major human pathogenic arenaviruses. Apart from being potent in acute infection, the drug is remarkably active in clearing chronic infection and potentiated the anti-arenaviral activity of Rib.

SwissDock, a protein-small molecule docking web service based on EADock DSS.

Most life science processes involve, at the atomic scale, recognition between two molecules. The prediction of such interactions at the molecular level, by so-called docking software, is a non-trivial task. Docking programs have a wide range of applications ranging from protein engineering to drug design. This article presents SwissDock, a web server dedicated to the docking of small molecules on target proteins. It is based on the EADock DSS engine, combined with setup scripts for curating common problems and for preparing both the target protein and the ligand input files. An efficient Ajax/HTML interface was designed and implemented so that scientists can easily submit dockings and retrieve the predicted complexes. For automated docking tasks, a programmatic SOAP interface has been set up and template programs can be downloaded in Perl, Python and PHP. The web site also provides an access to a database of manually curated complexes, based on the Ligand Protein Database. A wiki and a forum are available to the community to promote interactions between users. The SwissDock web site is available online at http://www.swissdock.ch. We believe it constitutes a step toward generalizing the use of docking tools beyond the traditional molecular modeling community.

Effect of Platinum-Based Chemoradiotherapy on Cellular Proliferation in Bone Marrow and Spleen, Estimated by 18F-FLT PET/CT in Patients with Locally Advanced Non-Small Cell Lung Cancer.

Historically, it has been difficult to monitor the acute impact of anticancer therapies on hematopoietic organs on a whole-body scale. Deeper understanding of the effect of treatments on bone marrow would be of great potential value in the rational design of intensive treatment regimens. 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) is a functional radiotracer used to study cellular proliferation. It is trapped in cells in proportion to thymidine-kinase 1 enzyme expression, which is upregulated during DNA synthesis. This study investigates the potential of (18)F-FLT to monitor acute effects of chemotherapy on cellular proliferation and its recovery in bone marrow, spleen, and liver during treatment with 2 different chemotherapy regimens.

Identification of small RNAs in Pseudomonas species

Summary: Bacterial small RNAs (sRNAs) are transcripts most of which have regulatory functions. Sequence and secondary structure elements enable numerous sRNAs to interact with mRNAs or with regulatory proteins resulting in diverse regulatory effects on virulence, iron storage, organization of cell envelope proteins or stress response. sRNAs having high affinity for RsmA-like RNA-binding proteins are important for posttranscriptional regulation in various Gram-negative bacteria. In Pseudomonas spp., the GacS/GacA two component system positively controls the production of such sRNAs. They titrate RsmA-like proteins and thus overcome translational repression due to these proteins. As a consequence, secondary metabolites can be produced that are implicated in the biocontrol capacity of P. fluorescens or in the virulence of P. aeruginosa. A genome-wide search carried out in P. aeruginosa PAO1 and in closely related Pseudomonas spp. resulted in the identification of 15 genes coding for sRNAs. Eight of these are novel, the remaining seven have previously been observed. Among them, the 1698 sRNA gene was expressed under GacA control, whereas the transcription of 1887 sRNA gene was transcribed under the control of the anaerobic regulator Anr in an oxygen-limited environment. Overexpression of 1698 sRNA in P. fluorescens strain CHAO did not affect the expression of the GacA-regulated hcnA gene (first gene of the operon coding for HCN synthase), indicating that 1698 sRNA is probably not part of the secondary metabolite regulation pathway. The expression of 1698 sRNA was positively regulated by RpoS in both P. aeruginosa PAO 1 and P. ,fluorescens CHAO and appeared to be modulated temporarily by oxidative stress conditions. However, the effect of 1698 sRNA on oxidative stress survival has not yet been established. Hfq protein interacted with 1698 sRNA in vitro and improved 1698 sRNA expression in vivo in P. aeruginosa. In P. fluorescens, GacA and Hfq were both required for expression of rpoS and GacA showed a positively control on the hfq expression; therefore, at least in this organism, GacA control of 1698 sRNA expression may act indirectly via Hfq and RpoS. Different methods were employed to find abase-pairing target for 1698 sRNA. In a proteomic analysis carried out in P. aeruginosa, positive regulation by 1698 sRNA was observed for Soda, the iron-associated superoxide dismutase, an enzyme involved in oxidative stress resistance. A sequence complementary with 1698 sRNA was predicted to be located in the 5' leader of soda mRNA. However, base-pairing between soda mRNA and 1698 sRNA remains to be proven. In conclusion, this work has revealed eight novel sRNAs and novel functions of two sRNAs in Pseudomonas spp. Résumé Les petits ARNs non-codants (sRNAs) produits par les bactéries sont des transcrits ayant pour la plupart des activités régulatrices importantes. Leurs séquences nucléotidiques ainsi que leurs structures secondaires permettent aux sRNAs d'interagir soit avec des RNA messagers (mRNAs), de sorte à modifier l'expression des protéines pour lesquelles ils codent, soit avec des protéines régulatrices liant des rnRNAs, ce qui a pour effet de modifier l'expression de ces mRNAs. Des sRNAs sont impliqués dans diverses voies de régulation, telles que celles qui régissent la virulence, le stockage du fer, l'organisation des protéines de l'enveloppe bactérienne ou la réponse au stress. Chez les Pseudomonas spp., le système à deux composantes GacS/GacA contrôle la production de métabolites secondaires. Ceux-ci sont engagés dans l'établissement du biocontrôle, chez P. fluorescens, ou. de la virulence, chez P. aeruginosa. La régulation génique dirigée par le système GacS/GacA fait intervenir les sRNAs du type RsmZ, capables de contrecarrer l'action au niveau traductionnel exercée par les protéines régulatrices du type RsmA. Une recherche au niveau du génome a été menée chez P. aeruginosa PAO1 de même que chez des espèces qui lui sont étroitement apparentées, débouchant sur la mise en évidence de 15 gènes codant pour des sRNAs. Parmi ceux-ci, huit ont été découverts pour la première fois et sept confirment des travaux publiés. L'expression du gène du sRNAs 1698 s'avère être régulée par GacA, vraisemblablement de manière indirecte. La transcription du gène du sRNA 1887 montre une dépendance envers Anr, régulateur de l'anaérobiose, et envers une carence en oxygène. La surexpression du sRNA 1698 chez P. fluorescens CHAO n'affecte pas l'expression de hcnA, un gène du régulon GacA, laissant supposer que le sRNA n'intervient pas dans la régulation des métabolites secondaires. Chez P. aeruginosa PAOI et chez P. fluorescens CHAO, RpoS, le facteur sigma du stress, est nécessaire à l'expression du sRNA 1698, et la concentration de ce dernier est modulée par des conditions de stress oxydatif. Toutefois, un effet du sRNA 1698 quant à la survie suite au stress oxydatif n'a pas été établi. Par ailleurs, l'interaction entre le sRNA 1698 et Hfq, la protéine chaperone de RNAs, in vitro ainsi qu'un rôle positif de Hfq pour l'expression du sRNA 1698 in vivo ont été démontrés chez P. aeruginosa. L'induction de l'expression par GacA de rpoS et de hfq a été confirmée chez P. fluorescens CHAO, suggérant que la régulation par GacA du sRNA 1698 pourrait se faire par l'intermédiaire de RpoS et Hfq. Diverses méthodes ont été employées pour identifier un transcrit qui puisse être apparié par le sRNA 1698. Une analyse de protéome chez P. aeruginosa montre que l'expression de Soda, la superoxyde dismutase associée au fer, est positivement régulée par le sRNA 1698. Soda est une enzyme impliquée dans la résistance au stress oxydatif. Une séquence de complémentarité avec le sRNA 1698 a bien été prédite sur le leader 5' du mRNA de soda. Cependant, l'appariement entre le sRNA et son transcrit cible est encore à prouver. En conclusion, ce travail a dévoilé huit nouveaux sRNAs et de nouvelles fonctions pour deux sRNAs chez les Pseudomonas.

Small RNAs as regulators of primary and secondary metabolism in Pseudomonas species.

Small RNAs (sRNAs) exert important functions in pseudomonads. Classical sRNAs comprise the 4.5S, 6S, 10Sa and 10Sb RNAs, which are known in enteric bacteria as part of the signal recognition particle, a regulatory component of RNA polymerase, transfer-messenger RNA (tmRNA) and the RNA component of RNase P, respectively. Their homologues in pseudomonads are presumed to have analogous functions. Other sRNAs of pseudomonads generally have little or no sequence similarity with sRNAs of enteric bacteria. Numerous sRNAs repress or activate the translation of target mRNAs by a base-pairing mechanism. Examples of this group in Pseudomonas aeruginosa are the iron-repressible PrrF1 and PrrF2 sRNAs, which repress the translation of genes encoding iron-containing proteins, and PhrS, an anaerobically inducible sRNA, which activates the expression of PqsR, a regulator of the Pseudomonas quinolone signal. Other sRNAs sequester RNA-binding proteins that act as translational repressors. Examples of this group in P. aeruginosa include RsmY and RsmZ, which are central regulatory elements in the GacS/GacA signal transduction pathway, and CrcZ, which is a key regulator in the CbrA/CbrB signal transduction pathway. These pathways largely control the extracellular activities (including virulence traits) and the selection of the energetically most favourable carbon sources, respectively, in pseudomonads.

Calculation of correction factors for ionization chamber measurements with small fields in low-density media.

The quantity of interest for high-energy photon beam therapy recommended by most dosimetric protocols is the absorbed dose to water. Thus, ionization chambers are calibrated in absorbed dose to water, which is the same quantity as what is calculated by most treatment planning systems (TPS). However, when measurements are performed in a low-density medium, the presence of the ionization chamber generates a perturbation at the level of the secondary particle range. Therefore, the measured quantity is close to the absorbed dose to a volume of water equivalent to the chamber volume. This quantity is not equivalent to the dose calculated by a TPS, which is the absorbed dose to an infinitesimally small volume of water. This phenomenon can lead to an overestimation of the absorbed dose measured with an ionization chamber of up to 40% in extreme cases. In this paper, we propose a method to calculate correction factors based on the Monte Carlo simulations. These correction factors are obtained by the ratio of the absorbed dose to water in a low-density medium □D(w,Q,V1)(low) averaged over a scoring volume V₁ for a geometry where V₁ is filled with the low-density medium and the absorbed dose to water □D(w,QV2)(low) averaged over a volume V₂ for a geometry where V₂ is filled with water. In the Monte Carlo simulations, □D(w,QV2)(low) is obtained by replacing the volume of the ionization chamber by an equivalent volume of water, according to the definition of the absorbed dose to water. The method is validated in two different configurations which allowed us to study the behavior of this correction factor as a function of depth in phantom, photon beam energy, phantom density and field size.

The European Respiratory Society spirometry tent: a unique form of screening for airway obstruction.

In order to raise public awareness of the importance of early detection of airway obstruction and to enable many people who had not been tested previously to have their lung function measured, the European Lung Foundation and the European Respiratory Society (ERS) organised a spirometry testing tent during the annual ERS Congresses in 2004-2009. Spirometry was performed during the ERS Congresses in volunteers; all participants answered a simple, brief questionnaire on their descriptive characteristics, smoking and asthma. Portable spirometers were freely provided by the manufacturer. Nurses and doctors from pulmonary departments of local hospitals/universities gave their service for free. Lower limit of normal (LLN) and Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria for diagnosing and grading airway obstruction were used. Of 12,448 participants in six congress cities, 10,395 (83.5%) performed acceptable spirometry (mean age 51.0 ± 18.4 yrs; 25.5% smokers; 5.5% asthmatic). Airway obstruction was present in 12.4% of investigated subjects according to LLN criteria and 20.3% according to GOLD criteria. Through multinomial logistic regression analysis, age, smoking habits and asthma were significant risk factors for airway obstruction. Relative risk ratio and 95% confidence interval for LLN stage I, for example, was 2.9 (2.0-4.1) for the youngest age (≤ 19 yrs), 1.9 (1.2-3.0) for the oldest age (≥ 80 yrs), 2.4 (2.0-2.9) for current smokers and 2.8 (2.2-3.6) for reported asthma diagnosis. In addition to being a useful advocacy tool, the spirometry tent represents an unusual occasion for early detection of airway obstruction in large numbers of city residents with an important public health perspective.

Interstitial cells of Cajal are normally distributed in both ganglionated and aganglionic bowel in Hirschsprung's disease

Résumé Introduction : La chirurgie de la maladie de Hirschsprung est fréquemment compliquée d'une atteinte post-opératoire de la motilité intestinale. Des anomalies du système nerveux entérique (SNE) telles que la dysplasie neuronale intestinale de type B, l'hypoganglionose ou l'aganglionose, présents dans le segment abaissé, peuvent être la cause de certaines de ces complications mais aucune information n'est disponible quant au rôle des cellules interstitielles de Cajal (CIC) sur la motilité intestinale dans la phase post-opératoire. Ces cellules sont considérées avoir un rôle de pacemaker dans le tractus gastro-intestinal. L'objectif de cette étude était de décrire la distribution des CIC dans le segment proximal du côlon réséqué lors de cures chirurgicales de maladie de Hirschsprung et de confronter ces observations à l'évolution clinique post-opératoire. Matériel et Méthodes : L'incidence des complications post-opératoires a été déterminée par une revue rétrospective des dossiers de 48 patients opérés pour maladie de Hirschspung entre 1977 et 1999 et par l'étude histologique et immuno-histochimique des pièces réséquées chez ces patients. Nous avons comparé la distribution des CIC dans le segment proximal du côlon avec celle du côlon sain de 16 enfants contrôles par microscopie optique. L'immunohistochimie au c-Kit a été utilisée pour marquer spécifiquement les CIC sur échantillons paraffinés. Ces résultats ont ensuite été corrélés avec l'étude du SNE de ces mêmes segments, déterminée par immunohistochimie au CD56 et au protein gene product 9.5. Résultats Les complications post-opératoires suivantes furent identifiées : constipation 46%, constipation avec incontinence 15%, entérocolite 8%, décès 4% (probablement sur entérocolite). La distribution des CIC dans les segments proximaux réséqués chez les enfants avec maladie de Hirschsprung était identique à celle observée dans les segments de côlon sain, et ce indépendamment de la distribution normale ou anormale du SNE. Chez les enfants opérés pour maladie de Hirschsprung les segments réséqués présentaient les anomalies d'innervation suivantes : aganglionose 10.4%, hypoganglionose 12.5%, dysplasie neuronale intestinale de type B 6.3%, autres dysganglionoses 14.6%. Aucune relation entre ces anomalies d'innervation et les complications post-opératoires n'a été mise en évidence. Conclusion : La distribution des CIC est normale chez les patient opérés pour maladie de Hirschsprung, et ne contribue donc pas aux atteintes post-opératoires de la motilité intestinale. Cela signifie aussi que le réseau de CIC se développe noinialement dans le côlon humain, même en présence d'une innervation colique anormale ou absente. Abstract: Surgery for Hirschsprung's disease is often complicated by post-operative bowel motility disorders. The impact of intestinal neural histology on the surgical outcome has been previously studied, but no information is available concerning the influence of the distribution of interstitial cells of Cajal (ICC) on these complications. These cells are considered to be pacemakers in the gastrointestinal tract. The aim of this study was to assess the distribution of ICC in the proximal segment of resected bowel in Hirschsprung's disease and confront these results with the clinical outcome. Using immunohistochemistry for light microscopy, we compared the pattern of distribution of ICC in the proximal segment of resected bowel in Hirschsprung's disease with that in normal colon. We correlated these results with the corresponding neural intestinal histology determined by CD56 and the protein gene product 9.5 immunohistochemistry. The distribution of ICC in the proximal segment of resected bowel is identical to that of normal colon, regardless of normal or abnormal colon innervation. ICC distribution does not seem to contribute to post-operative bowel motility disorders in patients operated for Hirschsprung's disease.

Oral epidermal growth factor receptor tyrosine kinase inhibitors for the treatment of non-small cell lung cancer: comparative pharmacokinetics and drug-drug interactions.

The development of orally active small molecule inhibitors of the epidermal growth factor receptor (EGFR) has led to new treatment options for non-small cell lung cancer (NSCLC). Patients with activating mutations of the EGFR gene show sensitivity to, and clinical benefit from, treatment with EGFR tyrosine kinase inhibitors (EGFR-TKls). First generation reversible ATP-competitive EGFR-TKls, gefitinib and erlotinib, are effective as first, second-line or maintenance therapy. Despite initial benefit, most patients develop resistance within a year, 50-60% of cases being related to the appearance of a T790M gatekeeper mutation. Newer, irreversible EGFR-TKls - afatinib and dacomitinib - covalently bind to and inhibit multiple receptors in the ErbB family (EGFR, HER2 and HER4). These agents have been mainly evaluated for first-line treatment but also in the setting of acquired resistance to first-generation EGFR-TKls. Afatinib is the first ErbB family blocker approved for patients with NSCLC with activating EGFR mutations; dacomitinib is in late stage clinical development. Mutant-selective EGFR inhibitors (AZD9291, CO-1686, HM61713) that specifically target the T790M resistance mutation are in early development. The EGFR-TKIs differ in their spectrum of target kinases, reversibility of binding to EGFR receptor, pharmacokinetics and potential for drug-drug interactions, as discussed in this review. For the clinician, these differences are relevant in the setting of polymedicated patients with NSCLC, as well as from the perspective of innovative anticancer drug combination strategies.