Recent research shows that electrostatic precipitation is a gentle method to collect airborne microorganisms and preserve their cultivability. However, the corona discharge used to charge the particles and the high electric field used to capture them are known to have a germicidal effect. The present paper investigates this paradoxical situation. Vegetative cells of E. coli and B. subtilis and spores of A. fumigatus and B. subtilis were deposited on different media and subjected to electrostatic fields of different strengths and polarities for controlled time periods. Vegetative cells are inactivated on cultivation agar plates, but remain cultivable when exposed on a stainless steel electrode and transferred afterwards onto agar plates. For the investigated conditions, spores were not affected by the corona discharge. Further experiments with a pH indicator show that chemical reactions occur when an aqueous media is exposed to the discharge. Some of these reactions are likely to create hydrogen peroxide which is known to kill a broad range of microorganisms. It is therefore highlighted that collecting electrodes in electrostatic air samplers should rather be dry conductive media.
Abstract Background Janus Kinases (JAK), including JAK1, JAK2, JAK3 and TYK2, have garnered increasing interest as therapeutic targets for several chronic inflammatory diseases such as inflammatory bowel disease (IBD). Although oral small molecule JAK inhibitors are being used in the clinics as an effective treatment for moderate-to-severe ulcerative colitis, nonethelessthey can lead to severe systemic immunosuppression, which is associated with an increased risk of infections. Our project aimed to develop a nanomedicine that induced JAK1 mRNA degradation within the intestinal mucosa with little systemic distribution. Methods The NEWDEAL project (H2020) developed both potent small interfering RNAs (siRNA) specific for JAK1 (siJAK1) or TNF (siTNF), and lipid nanoparticles (CL40) as nano-vectors. Their biodistribution in a mice colitis model was analyzed using labeled AF488-siJAK1 complexed to CL40 and given intrarectally (IR) for 3 consecutive days. In vivo cellular uptake was determined by FACS analysis. To induce colitis, C57/Bl6 mice received 2.5% dextrate sulfate sodium (DSS) in drinking water for 5 days. To determine efficacy of siRNAs, mice started DSS on day 0 and received IR administrations of siRNAs alone or complexed with CL40 nanoparticles on days -3, -2, -1, 1, 3, 5 and 7. On day 9, mice were sacrificed and disease activity index (DAI), histological score, fecal inflammatory markers and qPCR of targeted genes were analyzed. Results Colonic phagocytic cells - such as macrophages, neutrophils and monocytes - are the main targets cells of the siRNA-CL40 nanocomplexes following IR administration. A small percentage of cells in the draining lymph nodes captured siRNA CL40 nanocomplexes, remaining almost undetectable in spleen. This suggests a localized effect of the siRNA nanocomplexes after IR administration. While siJAK1-alone effectively reduced JAK1 mRNA expression in the distal colon, it did not improve intestinal inflammation induced by administering DSS. siTNF-CL40 treatment, though not siTNF-alone, significantly decreased intestinal inflammation in the DSS colitis model of treated mice, revealing significant protection from weight loss, lower histological scores, and reduced production of fecal lipocalin. Conclusion In summary, these results show that despite effectively reducing JAK1 mRNA expression, siJAK1 administration does not ameliorate colitis in a DSS mice model. However, we showed that a locally delivered siTNF-nanocomplex has the capacity to improve clinical, macroscopic and histological symptoms as well as fecal biomarkers in DSS colitic mice. We conclude not only that siRNA-CL40 target delivery is a good therapeutic strategy, but also that siTNF-CL40 is effective in locally treating intestinal inflammation.
Nous avons etudie les proprietes dynamiques de longues chaines d'adn dans le but de mieux comprendre leur comportement intracellulaire. Nous avons tout d'abord etudie les proprietes de solutions semi-diluees d'adn de bacteriophage t4. A faible gradient de cisaillement, la solution a un comportement newtonien. Les variations de la viscosite et du temps de reptation en fonction de la concentration sont identiques a celles obtenues avec des polymeres synthetiques. Pour des gradients de cisaillement eleves, la solution a des proprietes viscoelastiques non lineaires. Pour la concentration d'adn la plus elevee, la contrainte presente un plateau sur deux decades de gradient de cisaillement. Les adn topoisomerases sont des enzymes qui permettent de resoudre les problemes topologiques des molecules d'adn lors du cycle cellulaire. Un modele prevoit une modification du comportement dynamique de longues chaines d'adn lineaires en presence de ces enzymes. Nous avons tente de verifier les predictions du modele mais nos experiences ne permettent pas de conclure. Dans une seconde partie, nous avons etudie l'influence de la conformation de la chaine d'adn sur la reaction de cyclisation. Nous avons tout d'abord propose un diagramme decrivant la conformation de la chaine en fonction de la concentration en ions monovalents et en spermidine. L'etude de la reaction de cyclisation a ensuite montre des differences importantes lorsque la chaine passe de la conformation pelote a la conformation de globule : la cinetique est acceleree par un facteur 10#6, l'energie d'activation est diminuee par un facteur 3, les variations d'enthalpie et d'entropie sont plus importantes, la concentration effective est augmentee d'un facteur 9. 10#4. Enfin, nous avons etudie la region dans laquelle la reaction de cyclisation est la plus rapide (cyclisation en une seconde). La zone de maximum de cyclisation en une seconde semble correspondre a l'arrangement le plus dense possible de l'adn dans le globule.
Point-of-care (POC) systems require significant component integration to implement biochemical protocols associated with molecular diagnostic assays. Hybrid platforms where discrete components are combined in a single platform are a suitable approach to integration, where combining multiple device fabrication steps on a single substrate is not possible due to incompatible or costly fabrication steps. We integrate three devices each with a specific system functionality: (i) a silicon electro-wetting-on-dielectric (EWOD) device to move and mix sample and reagent droplets in an oil phase, (ii) a polymer microfluidic chip containing channels and reservoirs and (iii) an aqueous phase glass microarray for fluorescence microarray hybridization detection. The EWOD device offers the possibility of fully integrating on-chip sample preparation using nanolitre sample and reagent volumes. A key challenge is sample transfer from the oil phase EWOD device to the aqueous phase microarray for hybridization detection. The EWOD device, waveguide performance and functionality are maintained during the integration process. An on-chip biochemical protocol for arrayed primer extension (APEX) was implemented for single nucleotide polymorphism (SNiP) analysis. The prepared sample is aspirated from the EWOD oil phase to the aqueous phase microarray for hybridization. A bench-top instrumentation system was also developed around the integrated platform to drive the EWOD electrodes, implement APEX sample heating and image the microarray after hybridization.
Resumen del trabajo presentado a la VIII National Conference BIFI, celebrada en el Edificio I+D Campus Rio Ebro de la Universidad de Zaragoza, del 31 de enero al 2 de febrero de 2017.
Most procedures for detecting pathogens in liquid media require an initial concentration step. In this regard, carbohydrates have proven to be attractive affinity ligands for the solid-phase capture of bacteria that use lectins for adhesion to host cell membranes. However, the use of cyclodextrin-immobilized substrates to selectively trap bacteria has not been explored before. Here, using quartz-crystal microbalance with dissipation monitoring experiments, we demonstrate that functionalization of surfaces by β-cyclodextrin (β-CD) can not only allow for rapid and efficient capture of bacterial cells in liquid but also their facile elution with an aqueous solution of a selectively methylated β-CD derivative as a competitive molecule. This capture/elution strategy, which is based on host-guest interactions between membrane components of the bacterial cell and the CD cavities, is performed in physiological conditions and can be integrated in a microchip. Indeed, proof-of-concept studies showed the potential of β-CD-modified micropillar-integrated microfluidic devices for concentration of bacteria. The results obtained with Escherichia coli suggest that this approach could be broadly applicable among Gram-negative bacteria, which share common cell membrane structures.
Abstract Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.
Abstract Background Oral inhibitors of JAK1 have become promising therapeutic agents for the treatment of inflammatory bowel diseases (IBD); however, concerns have been raised regarding their specificity and safety profiles. Currently, a local therapy based on specific JAK1 siRNA combined with lipid nanoparticle (LNP) technology is under investigation as a safer alternative to JAK inhibitors.The purpose of this study is to explore the inhibition of the JAK1 pathway in the intestinal epithelium mediated by siRNA/LNP technology, using human primary 2D culture. Methods Human primary 2D cultures were generated from colonic 3D organoids of non-IBD donors. The efficiency of JAK1 pathway inhibition was tested in IFN-y stimulated cultures using either filgotinib (a JAK1 inhibitor, used as a control) or the novel human JAK1 siRNA. JAK1 siRNA transfection was performed using Lipofectamine or LNPs. qPCR was performed on a panel of JAK1 target genes to evaluate the efficiency of JAK1 pathway inhibition. Results Incubation of the 2D culture with IFN-y induced the activation of the JAK1 pathway, as suggested by the significant up-regulation of JAK1-dependent genes (i.e., CXCL10, SOCS1, SOCS3 and PLA2G2A). The addition of filgotinib to the culture efficiently inhibited the JAK1 pathway by suppressing the expression of JAK1-target genes. JAK1 siRNA transfection using Lipofectamine reduced JAK1 mRNA expression by 50%, which was mirrored by the concomitant down-regulation (between 60 and 80%) of JAK1-dependent genes. Importantly, the silencing efficiency of the JAK1-dependent pathway by LNPs was comparable to that observed using Lipofectamine. Conclusion Organoid-derived 2D culture is a useful model for investigating the activation of the JAK1 pathway and its pharmacological inhibition in human intestinal epithelium. In particular, siRNA/LNP nanoplexes may be a promising technology for locally delivering highly specific siRNAs to the intestinal mucosa, which could pave the way for the development of more effective treatments for IBD patients.
