Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells

Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement – a stage between initial cellular internalization and final gene silencing of siRNA delivery systems

The phenology of winter rye in Poland: an analysis of long-term experimental data

The study of the phenology of crops, although quite popular, has limitations, mainly because of frequent changes to crop varieties and management practices. Here, we present data on the phenology and yield of winter rye in western Poland collected between 1957 and 2012 from a long-term field experiment. Data were examined for trends through time and compared to climatological factors using regression analysis. Both annual air temperature and precipitation increased during the study period, equivalent to 2 °C and 186 mm, respectively, over the 52-year period for which met data were available. We detected significant delays in sowing date and recently in emergence, but significant advances were apparent in full flowering date equivalent to 4 days/decade. Yield and plant density experienced a step like change in 1986; yield increasing by ca. 70 % and plant density increasing by ca. 50 %, almost coinciding with a similar change in annual mean temperature, but most likely caused by a changed seed rate and use of herbicides. Future climate change is expected to have a greater impact on this crop, but farmers may be able to adapt to these changes by modifying water regimes, using new machinery and sowing new rye varieties

Spatial and Temporal Variations in the Annual Pollen Index Recorded by Sites Belonging to the Portuguese Aerobiology Network

This study presents the findings of a 10-year survey carried out by the Portuguese Aerobiology Network (RPA) at seven pollen-monitoring stations: five mainland stations (Oporto, Coimbra, Lisbon, Évora and Portimão) and two insular stations [Funchal (Madeira archipelago) and Ponta Delgada (Azores archipelago)]. The main aim of the study was to examine spatial and temporal variations in the Annual Pollen Index (API) with particular focus on the most frequently recorded pollen types. Pollen monitoring (2003–2012) was carried out using Hirst-type volumetric spore traps, following the minimum recommendations proposed by the European Aerobiology Society Working Group on Quality Control. Daily pollen data were examined for similarities using the Kruskal–Wallis nonparametric test and multivariate regression trees. Simple linear regression analysis was used to describe trends in API. The airborne pollen spectrum at RPA stations is dominated by important allergenic pollen types such as Poaceae, Olea and Urticaceae. Statistically significant differences were witnessed in the API recorded at the seven stations. Mean API is higher in the southern mainland cities, e.g. Évora, Lisbon and Portimão, and lower in insular and littoral cities. There were also a number of significant trends in API during the 10-year study. This report identifies spatial and temporal variations in the amount of airborne pollen recorded annually in the Portuguese territory. There were also a number of significant changes in API, but no general increases in the amount of airborne pollen

Dynamic Measurements of Membrane Insertion Potential of Synthetic Cell Penetrating Peptides

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/la403370p.Cell penetrating peptides (CPPs) have been established as excellent candidates for mediating drug delivery into cells. When designing synthetic CPPs for drug delivery applications, it is important to understand their ability to penetrate the cell membrane. In this paper, anionic or zwitterionic phospholipid monolayers at the air-water interface are used as model cell membranes to monitor the membrane insertion potential of synthetic CPPs. The insertion potential of CPPs having different cationic and hydrophobic amino acids were recorded using a Langmuir monolayer approach that records peptide adsorption to model membranes. Fluorescence microscopy was used to visualize alterations in phospholipid packing due to peptide insertion. All CPPs had the highest penetration potential in the presence of anionic phospholipids. In addition, two of three amphiphilic CPPs inserted into zwitterionic phospholipids, but none of the hydrophilic CPPs did. All the CPPs studied induced disruptions in phospholipid packing and domain morphology, which were most pronounced for amphiphilic CPPs. Overall, small changes to amino acids and peptide sequences resulted in dramatically different insertion potentials and membrane reorganization. Designers of synthetic CPPs for efficient intracellular drug delivery should consider small nuances in CPP electrostatic and hydrophobic properties

Polysialic acid sustains cancer cell survival and migratory capacity in a hypoxic environment

Polysialic acid (polySia) is a unique carbohydrate polymer expressed on the surface of NCAM (neuronal cell adhesion molecule) in a number of cancers where it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis and is strongly associated with poor clinical prognosis. We have carried out the first investigation into the effect of polySia expression on the behaviour of cancer cells in hypoxia, a key source of chemoresistance in tumours. The role of polysialylation and associated tumour cell migration and cell adhesion were studied in hypoxia, along with effects on cell survival and the potential role of HIF-1. Our findings provide the first evidence that polySia expression sustains migratory capacity and is associated with tumour cell survival in hypoxia. Initial mechanistic studies indicate a potential role for HIF-1 in sustaining polySia-mediated migratory capacity, but not cell survival. These data add to the growing body of evidence pointing to a crucial role for the polysialyltransferases (polySTs) in neuroendocrine tumour progression and provide the first evidence to suggest that polySia is associated with an aggressive phenotype in tumour hypoxia. These results have significant potential implications for polyST inhibition as an anti-metastatic therapeutic strategy and for targeting hypoxic cancer cells

Detrusor bioengineering using a cell‐enriched compressed collagen hydrogel

OBJECTIVE: The gold standard for bladder regeneration in end-stage bladder disease is the use of intestinal tissue, which is however associated with significant long-term complications. Our study aims to bioengineer functional detrusor muscle combining bladder smooth muscle cells (SMC) and SMC-like adipose-derived stem cells (pADSC) in compressed collagen (CC) hydrogels and to investigate biocompatibility and tissue regeneration of such detrusor-equivalents in a rat detrusorectomy model. METHODS: Compressed collagen hydrogels seeded with 1 × 10$^{6}$ or 4 × 10$^{6}$ SMC alone or in combination with pADSC in a 1:1 ratio were investigated. Morphology, phenotype, and viability as well as proteomic secretome analysis were assessed in the 1:1 co-cultures and the respective monocultures. The hydrogels were implanted into rat bladders after partial detrusorectomy. Bladders were harvested 8 weeks after transplantation, and assessed for tissue morphology, detrusor regeneration, neo-vascularization and -innervation. RESULTS: Co-cultured cells exhibited native SMC morphology, high viability and proliferated to form microtissues in vitro. The pro-angiogenic factors angiogenin, vascular endothelial growth factor (VEGF)-A and -D were increased in the secretome of the pADSC samples. After 8 weeks of in vivo, the regenerated bladder wall showed a multilayered structure containing all bladder wall components. The overall performance of the bladder wall regeneration of CC seeded with 4 × 10$^{6}$ cells was significantly better than with 1 × 10$^{6}$ cells and the combination SMC:pADCS performed slightly better than SMC alone. CONCLUSION: Compressed collagen possesses an adequate regenerative potential to promote regeneration of bladder wall tissue in vivo. Seeded with a combination of pADSC and SMC this may well be the first step towards a functional bladder reconstruction especially in patients suffering of end-stage bladder diseases