Dendrimer-coated carbon nanotubes deliver dsRNA and increase the efficacy of gene knockdown in the red flour beetle Tribolium castaneum.

In this study, the use of dendrimer-coated carbon nanotubes (CNTs) as a delivery vehicle for dsRNA was assessed in Tribolium castaneum. Exposure to low dosages of polyamidoamine dendrimer carbon nanotubes (PAMAM-CNTs) did not affect T. castaneum larval mortality. Expression of key apoptotic factors, Dronc (Tc12580), Dredd (Tcn-like, Tc014026) and Buffy, (Tcinhib apop1), which can act as toxicity indicators, were not altered in T. castaneum larvae following injection of PAMAM-CNTs. The level of knockdown of two target genes, α-tubulin and mitochondrial RNA polymerase (mtpol), were significantly increased when larvae were injected with double-stranded RNA bound to CNTs (PAMAM-CNT-dsRNA), compared to those injected with target dsRNA alone. PAMAM-CNTs were visualised in cellular vacuoles and in the cell nucleus. Increase occurrence of a blistered wing phenotype was found in a subset of PAMAM-CNT-dsRNAαtub injected larvae, relative to the level seen in larvae injected with naked dsRNAαtub alone. These results suggest that the use of functionalised CNTs for dsRNA delivery could increase the efficacy of RNA interference in insect pest species.

Biophysical Characterization of Membrane Phase Transition Profiles for the Discrimination of Outer Membrane Vesicles (OMVs) From Escherichia coli Grown at Different Temperatures.

Dynamic Light Scattering (DLS), Small Angle X-ray Scattering (SAXS) and Transmission Electron Microscopy (TEM) are physical techniques widely employed to characterize the morphology and the structure of vesicles such as liposomes or human extracellular vesicles (exosomes). Bacterial extracellular vesicles are similar in size to human exosomes, although their function and membrane properties have not been elucidated in such detail as in the case of exosomes. Here, we applied the above cited techniques, in synergy with the thermotropic characterization of the vesicles lipid membrane using a turbidimetric technique to the study of vesicles produced by Gram-negative bacteria (Outer Membrane Vesicles, OMVs) grown at different temperatures. This study demonstrated that our combined approach is useful to discriminate vesicles of different origin or coming from bacteria cultured under different experimental conditions. We envisage that in a near future the techniques employed in our work will be further implemented to discriminate complex mixtures of bacterial vesicles, thus showing great promises for biomedical or diagnostic applications.

Biophysical and biological contributions of polyamine-coated carbon nanotubes and bidimensional buckypapers in the delivery of miRNAs to human cells.

Recent findings in nanomedicine have revealed that carbon nanotubes (CNTs) can be used as potential drug carriers, therapeutic agents and diagnostics tools. Moreover, due to their ability to cross cellular membranes, their nanosize dimension, high surface area and relatively good biocompatibility, CNTs have also been employed as a novel gene delivery vector system. In our previous work, we functionalized CNTs with two polyamine polymers, polyethyleneimine (PEI) and polyamidoamine dendrimer (PAMAM). These compounds have low cytotoxicity, ability to conjugate microRNAs (such as miR-503) and, at the same time, transfect efficiently endothelial cells. The parameters contributing to the good efficiency of transfection that we observed were not investigated in detail. In fact, the diameter and length of CNTs are important parameters to be taken into account when evaluating the effects on drug delivery efficiency. In order to investigate the biophysical and biological contributions of polymer-coated CNTs in delivery of miRNAs to human cells, we decided to investigate three different preparations, characterized by different dimensions and aspect ratios. In particular, we took into account very small CNTs, a suspension of CNTs starting from the commercial product and a 2D material based on CNTs (ie, buckypapers [BPs]) to examine the transfection efficiency of a rigid scaffold. In conclusion, we extensively investigated the biophysical and biological contributions of polyamine-coated CNTs and bidimensional BPs in the delivery of miRNAs to human cells, in order to optimize the transfection efficiency of these compounds to be employed as efficient drug delivery vectors in biomedical applications.

How Our Other Genome Controls Our Epi-Genome.

Eukaryotes and prokaryotes produce extracellular nanovescicles that contain RNAs and other molecules that they exploit to communicate. Recently, inter-kingdom crosstalk was demonstrated between humans and bacteria through fecal microRNAs. We suggest here how bacteria interact with humans via RNAs within membrane vesicles to alter our epigenome, thus filling the gap and closing the circle. At the same time, there are indications that there could be a wider inter-kingdom communication network that might encompass all known kingdoms. Now that the connection with our other genome has been established, we also should begin to explore the 'social' network that we have around us.

Regulation of angiogenesis through the efficient delivery of microRNAs into endothelial cells using polyamine-coated carbon nanotubes.

MicroRNAs (miRNAs) directly regulate gene expression at a post-transcriptional level and represent an attractive therapeutic target for a wide range of diseases. Here, we report a novel strategy for delivering miRNAs to endothelial cells (ECs) to regulate angiogenesis, using polymer functionalized carbon nanotubes (CNTs). CNTs were coated with two different polymers, polyethyleneimine (PEI) or polyamidoamine dendrimer (PAMAM), followed by conjugation of miR-503 oligonucleotides as recognized regulators of angiogenesis. We demonstrated a reduced toxicity for both polymer-coated CNTs, compared with pristine CNTs or polymers alone. Moreover, polymer-coated CNT stabilized miR-503 oligonucleotides and allowed their efficient delivery to ECs. The functionality of PAMAM-CNT-miR-503 complexes was further demonstrated in ECs through regulation of target genes, cell proliferation and angiogenic sprouting and in a mouse model of angiogenesis. This comprehensive series of experiments demonstrates that the use of polyamine-functionalized CNTs to deliver miRNAs is a novel and effective means to regulate angiogenesis.

Keppen-Lubinsky syndrome is caused by mutations in the inwardly rectifying K+ channel encoded by KCNJ6.

Keppen-Lubinsky syndrome (KPLBS) is a rare disease mainly characterized by severe developmental delay and intellectual disability, microcephaly, large prominent eyes, a narrow nasal bridge, a tented upper lip, a high palate, an open mouth, tightly adherent skin, an aged appearance, and severe generalized lipodystrophy. We sequenced the exomes of three unrelated individuals affected by KPLBS and found de novo heterozygous mutations in KCNJ6 (GIRK2), which encodes an inwardly rectifying potassium channel and maps to the Down syndrome critical region between DIRK1A and DSCR4. In particular, two individuals shared an in-frame heterozygous deletion of three nucleotides (c.455_457del) leading to the loss of one amino acid (p.Thr152del). The third individual was heterozygous for a missense mutation (c.460G>A) which introduces an amino acid change from glycine to serine (p.Gly154Ser). In agreement with animal models, the present data suggest that these mutations severely impair the correct functioning of this potassium channel. Overall, these results establish KPLBS as a channelopathy and suggest that KCNJ6 (GIRK2) could also be a candidate gene for other lipodystrophies. We hope that these results will prompt investigations in this unexplored class of inwardly rectifying K(+) channels.

Aged iPSCs display an uncommon mitochondrial appearance and fail to undergo in vitro neurogenesis.

Reprogramming of human fibroblasts into induced pluripotent stem cells (iPSCs) leads to mitochondrial rejuvenation, making iPSCs a candidate model to study the mitochondrial biology during stemness and differentiation. At present, it is generally accepted that iPSCs can be maintained and propagated indefinitely in culture, but no specific studies have addressed this issue. In our study, we investigated features related to the 'biological age' of iPSCs, culturing and analyzing iPSCs kept for prolonged periods in vitro. We have demonstrated that aged iPSCs present an increased number of mitochondria per cell with an altered mitochondrial membrane potential and fail to properly undergo in vitro neurogenesis. In aged iPSCs we have also found an altered expression of genes relevant to mitochondria biogenesis. Overall, our results shed light on the mitochondrial biology of young and aged iPSCs and explore how an altered mitochondrial status may influence neuronal differentiation. Our work suggests to deepen the understanding of the iPSCs biology before considering their use in clinical applications.