Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration

During the last years the potential role of in vitro transcribed (IVT) mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE). A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000) in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages of IVT mRNA as compared to pDNA as transfection agent.     

An Upregulation in the Expression of Vanilloid Transient Potential Channels 2 Enhances Hypotonicity-Induced Cytosolic Ca2+ Rise in Human Induced Pluripotent Stem Cell Model of Hutchinson Gillford Progeria

Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca²⁺ ([Ca²⁺]_i) rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca²⁺]_i rise in iPSC-ECs from normal individuals but a sustained [Ca²⁺]_i elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM), and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca²⁺]_i rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca²⁺]_i rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca²⁺]_i elevation in HGPS-iPSC-ECs under hypotonicity, consequently resulting in apoptotic cell death. This mechanism may contribute to the pathogenesis of vascular diseases in HGPS patients.     

Underwater locomotion strategy by a benthic pennate diatom Navicula sp.

The mechanism of diatom locomotion has been widely researched but still remains a hypothesis. There are several questionable points on the prevailing model proposed by Edgar, and some of the observed phenomena cannot be completely explained by this model. In this paper, we undertook detailed investigations of cell structures, locomotion, secreted mucilage, and bending deformation for a benthic pennate diatom Navicula species. According to these broad evidences, an updated locomotion model is proposed. For Navicula sp., locomotion is realized via two or more pseudopods or stalks protruded out of the frustules. The adhesion can be produced due to the pull-off of one pseudopod or stalk from the substratum through extracellular polymeric substances. And the positive pressure is generated to balance the adhesion because of the push-down of another pseudopod or stalk onto the substratum. Because of the positive pressure, friction is generated, acting as a driving force of locomotion, and the other pseudopod or stalk can detach from the substratum, resulting in the locomotion. Furthermore, this model is validated by the force evaluation and can better explain observed phenomena. This updated model would provide a novel aspect on underwater locomotion strategy, hence can be useful in terms of artificial underwater locomotion devices.     

Saltation of Non-Spherical Sand Particles

Saltation is an important geological process and the primary source of atmospheric mineral dust aerosols. Unfortunately, no studies to date have been able to precisely reproduce the saltation process because of the simplified theoretical models used. For example, sand particles in most of the existing wind sand movement models are considered to be spherical, the effects of the sand shape on the structure of the wind sand flow are rarely studied, and the effect of mid-air collision is usually neglected. In fact, sand grains are rarely round in natural environments. In this paper, we first analyzed the drag coefficients, drag forces, and starting friction wind speeds of sand grains with different shapes in the saltation process, then established a sand saltation model that considers the coupling effect between wind and the sand grains, the effect of the mid-air collision of sand grains, and the effect of the sand grain shape. Based on this model, the saltation process and sand transport rate of non-spherical sand particles were simulated. The results show that the sand shape has a significant impact on the saltation process; for the same wind speed, the sand transport rates varied for different shapes of sand grains by as much as several-fold. Therefore, sand shape is one of the important factors affecting wind-sand movement.     

A Novel Manno-Oligosaccharide Binding Protein Identified in Alkaliphilic Bacillus sp. N16-5 Is Involved in Mannan Utilization

ManH, a novel substrate-binding protein of an ABC transporter, was identified from the mannan utilization gene cluster of Bacillus sp. N16-5. We cloned, overexpressed, and purified ManH and measured its binding affinity to different substrates by isothermal titration calorimetry. ManH binds to mannotriose, mannotetraose, mannopentose, and galactosyl-mannotriose with dissociation constants in the micromolar range. Deletion of manH led to decreased growth ability of the strain when cultivated in medium with manno-oligosaccharides or mannan as the carbon source. ManH belongs to a manno-oligosaccharide transporter and plays an important role in mannan utilization by Bacillus sp. N16-5.