Use of inert gas jets to measure the forces required for mechanical gene transfection

BACKGROUND: Transferring genes and drugs into cells is central to how we now study, identify and treat diseases. Several non-viral gene therapy methods that rely on the mechanical disruption of the plasma membrane have been proposed, but the success of these methods has been limited due to a lack of understanding of the mechanical parameters that lead to cell membrane permeability. METHODS: We use a simple jet of inert gas to induce local transfection of plasmid DNA both in vitro (HeLa cells) and in vivo (chicken chorioallantoic membrane). Five different capillary tube inner diameters and three different gases were used to treat the cells to understand the dependency of transfection efficiency on the dynamic parameters. RESULTS: The simple setup has the advantage of allowing us to calculate the forces acting on cells during transfection. We found permeabilization efficiency was related to the dynamic pressure of the jet. The range of dynamic pressures that led to transfection in HeLa cells was small (200 ± 20 Pa) above which cell stripping occurred. We determined that the temporary pores allow the passage of dextran up to 40 kDa and reclose in less than 5 seconds after treatment. The optimized parameters were also successfully tested in vivo using the chorioallantoic membrane of the chick embryo. CONCLUSIONS: The results show that the number of cells transfected with the plasmid scales with the dynamic pressure of the jet. Our results show that mechanical methods have a very small window in which cells are permeabilized without injury (200 to 290 Pa). This simple apparatus helps define the forces needed for physical cell transfection methods

Microfluidic traction force microscopy to study mechanotransduction in angiogenesis.

The formation of new blood vessels from existing vasculature, angiogenesis, is driven by coordinated endothelial cell migration and matrix remodelling in response to local signals. Recently, a growing body of evidence has shown that mechanotransduction, along with chemotransduction, is a major regulator of angiogenesis. Mechanical signals, such as fluid shear stress and substrate mechanics, influence sprouting and network formation, but the mechanisms behind this relationship are still unclear. Here, we present cellular traction forces as possible effectors activated by mechanosensing to mediate matrix remodelling, and encourage the use of traction force microscopy to study mechanotransduction in angiogenesis. We also suggest that deciphering the response of endothelial cells to mechanical signals could reveal an optimal angiogenic mechanical environment, and provide insight into development, wound healing, the initiation and growth of tumours, and new strategies for tissue engineering. This article is protected by copyright. All rights reserved

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Future directions for therapeutic strategies in post-ischaemic vascularization: a position paper from European Society of Cardiology Working Group on Atherosclerosis and Vascular Biology

Modulation of vessel growth holds great promise for treatment of cardiovascular disease. Strategies to promote vascularization can potentially restore function in ischaemic tissues. On the other hand, plaque neovascularization has been shown to associate with vulnerable plaque phenotypes and adverse events. The current lack of clinical success in regulating vascularization illustrates the complexity of the vascularization process, which involves a delicate balance between pro- and anti-angiogenic regulators and effectors. This is compounded by limitations in the models used to study vascularization that do not reflect the eventual clinical target population. Nevertheless, there is a large body of evidence that validate the importance of angiogenesis as a therapeutic concept. The overall aim of this Position Paper of the ESC Working Group of Atherosclerosis and Vascular biology is to provide guidance for the next steps to be taken from pre-clinical studies on vascularization towards clinical application. To this end, the current state of knowledge in terms of therapeutic strategies for targeting vascularization in post-ischaemic disease is reviewed and discussed. A consensus statement is provided on how to optimize vascularization studies for the identification of suitable targets, the use of animal models of disease, and the analysis of novel delivery methods

Spatially restricted gene transfection using an inert gas and straight capillary tubes

Though mechanical means of gene transfection have many advantages, they are generally of low efficiency due to a lack of understanding of the underlying principles. We used a novel spatially restricted transfection technique in an attempt to gain significant insight into the underlying mechanisms of mechanical transfection methods. We demonstrate that a jet of inert gas flowing out of a straight capillary tube can facilitate local transfection of plasmid DNA both in vitro and in vivo. We show that there is a fine balance between transfection and cell death. Previous to this work, Leduc (2011) had used five different capillary tube inner diameters and three different gases to treat HeLa cells to understand the relationship between the transfection efficiency to the area covered by the gas jet, flow rate and properties of the different gases used (density and viscosity). He found temporary permeabilization to be specific to the path treated by the jet and that the efficiency was best correlated to the dynamic pressure of the jet. We extended the work to show that the range of dynamic pressures that led to transfection was small, in both HeLa and HAAEC adherent monolayer of cells (approximately 300 Pa). Following his findings, we imaged the transfection phenomenon using a 10 kDa dextran solution to evaluate the transfection efficiency (which peaked at 450 Pa at 73 ± 5%), along with stripping and cell death through Trypan-blue staining and confocal microscopy. We found a similar window of permeabilization, above which permanent permeabilization (cell death) and stripping of the cells occurred. These findings were reproduced in the HAAEC line. We then confirmed that the transient pores created allowed the passage of dextrans up to 40 kDa through confocal microscopy. Using Leduc's (2011) finding that the pores reclose in less than 5 seconds after treatment, we were able to fix an adherent cell line rapidly enough to image the pores. An SEM technique was developed to image the density and diameter of the pores, based on different dynamic pressures, created in HeLa and HAAEC. The transfection technique was also successfully used in vivo in the chorioallantoic membrane (CAM) of 7-day old chick embryos. Hence, temporary permeabilization of adherent cells and the CAM can be achieved by treating the biological tissue with an inert jet of gas.Bien que les méthodes mécaniques de transfert de gènes présentent de nombreux avantages, elles sont généralement peu efficaces en raison d'un manque de compréhension des principes sous-jacents. Nous avons donc développé une nouvelle technique simple de transfert de gènes, spatialement restreint, dans le but d'élucider certains des mécanismes sous-jacents de plusieurs méthodes de transfert des gènes mécaniques. Nous démontrons qu'il est possible d'utiliser un appareil simple construit à partir d'un capillaire rectiligne ancré à un tube de polymère, laissant pénétrer un jet de gaz inerte pour faciliter la transfection localisée d'ADN plasmidique à la fois dans un modèle in vitro et in vivo. Nous démontrons aussi qu'il existe un équilibre délicat entre la transfection et la mort cellulaire. Dans une étude antérieure, Leduc (2011) a utilisé cinq différents diamètres internes de tubes capillaires et trois gaz inertes différents pour traiter des cellules HeLa, en espérant pouvoir mieux comprendre la relation entre l'efficacité du transfert d'ADN (sur la surface couverte par le jet de gaz) et les propriétés des différents gaz utilisés (densité et viscosité). Nous avons étendu le travail de Leduc (2011) en confirmant que la gamme de pressions dynamiques qui conduit à la transfection transitoire est mince (environ 300 Pa), à la fois dans les cellules adhérentes HeLa et HAAEC cultivées en monocouche. Suite à ces constatations, nous avons photographié: le phénomène de transfection à l'aide d'une solution de dextran 10 kDa pour évaluer l'efficacité de la transfection (qui a culminé à 450 Pa à 73 ± 5% d'efficacité), la mort cellulaire et le décapage de cellules avec l'aide d'un microscope confocal et une solution de bleu de Trypan dans les cellules HeLa. Nous avons découvert qu'à partir de 300 Pa de pression dynamique le décapage et la perméabilisation permanente (mort cellulaire) devenaient substantielles. Ces résultats ont été reproduits dans la lignée HAAEC. Nous avons ensuite confirmé que les pores transitoires créés permettaient le passage de molécules de dextrans jusqu'à 40 kDa de taille par microscopie confocale. En se basant sur la découverte de Leduc (2011), selon laquelle les pores se referment en moins de 5 secondes après le traitement, nous avons été en mesure de fixer chimiquement une lignée de cellules adhérentes assez rapidement pour pouvoir les photographier par microscopie électronique à balayage (MEB). Une nouvelle technique de MEB a été développée pour photographier la densité et le diamètre des pores, sur des spécimens soumis à différentes pressions dynamiques, en utilisant des cellules HeLa et HAAEC. La technique de transfection a été également utilisée avec succès in vivo sur la membrane chorio-allantoïde (CAM) d'embryons de poulets âgés de sept jours. Pour conclure, la perméabilisation temporaire de cellules adhérentes et la CAM peut être facilitée grâce à un traitement simple de jet de gaz inerte

Pest Activity and Protection Practices: Four Decades of Transformation in Quebec Apple Orchards

A group of commercial orchards from Quebec (Canada) was followed from 1977 to 2019 as part of a project to implement Integrated Pest Management (IPM) practices. Collected data comprised activity of major fruit pests (from monitoring traps), fruit damage at harvest and pesticide applications, from which the annual costs and impacts of protection programs over 42 years were calculated. Activity and fruit damage in commercial orchards were compared to patterns observed in a reference insecticide-free orchard. Some insects (European apple sawfly, codling moth, apple maggot) were more prevalent in the insecticide-free orchard than in commercial orchards, while others were more prevalent in commercial orchards (oblique-banded leafroller) or as prevalent in both orchard types (tarnished plant bug). Annual fruit damage in the insecticide-free orchard was mostly from the apple maggot (up to 98%), the plum curculio (up to 90%) and the codling moth (up to 58%). The average situation was different in commercial orchards, whose damage was mostly from the plum curculio (up to 7.6%), the tarnished plant bug (up to 7.5%) and the oblique-banded leafroller (up to 1.7%). While the number of registered pesticides, the number of applications and the total cost of pesticides gradually increased from 2002 to 2019, the risks incurred, as measured by indicators of environmental and health impacts, followed a downward trend for insecticides and acaricides and varied slightly for fungicides

Increased shear stress inhibits angiogenesis in veins and not arteries during vascular development

Vascular development is believed to occur first by vasculogenesis followed by angiogenesis. Though angiogenesis is the formation of new vessels, we found that vascular density actually decreases during this second stage. The onset of the decrease coincided with the entry of erythroblasts into circulation. We therefore measured the level of shear stress at various developmental stages and found that it was inversely proportional to vascular density. To investigate whether shear stress was inhibitory to angiogenesis, we altered shear stress levels either by preventing erythroblasts from entering circulation ("low" shear stress) or by injection of a starch solution to increase the blood plasma viscosity ("high" shear stress). By time-lapse microscopy, we show that reverse intussusception (merging of two vessels) is inversely proportional to the level of shear stress. We also found that angiogenesis (both sprouting and splitting) was inversely proportional to shear stress levels. These effects were specific to the arterial or venous plexus however, such that the effect on reverse intussusception was present only in the arterial plexus and the effect on sprouting only in the venous plexus. We cultured embryos under altered shear stress in the presence of either DAPT, a Notch inhibitor, or DMH1, an inhibitor of the bone morphogenetic protein (BMP) pathway. DAPT treatment phenocopied the inhibition of erythroblast circulation ("low" shear stress) and the effect of DAPT treatment could be partially rescued by injection of starch. Inhibition of the BMP signaling prevented the reduction in vascular density that was observed when starch was injected to increase shear stress levels.status: publishe

GESTION DES DÉBITS D’UN BARRAGE EN MILIEU URBANISÉ

Le barrage Joseph-Samson est situé à l’embouchure de la rivière Saint-Charles au confluent du fleuve Saint-Laurent dans la Basse-Ville de Québec. Il crée un plan d’eau sur environ 3,7 km et avec ses clapets anti-marées, il protège la Basse-Ville des très grandes marées vives dans le fleuve. Cependant, le barrage a complètement changé un estuaire naturel en canal de sédimentation de matières provenant du bassin versant forestier, semi-urbain et urbain. Le libre passage des poissons est entravé et une réduction importante d’oxygène dissous est souvent observée. Le barrage pourrait contribuer aux risques d’inondation lorsque le débit de la rivière est élevé. Des simulations 1D (HEC-RAS) ont été réalisées pour évaluer l’impact de la présence du barrage sur les risques d’inondation pour différents scénarios d’ouverture et fermeture de sa vanne de fond. Des simulations 2D (River2D) ont aussi été réalisées pour évaluer l’impact du barrage sur les vitesses d’écoulement.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author