Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in Solanum species.

Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on Solanum crops and wild relatives. The methods that we further elaborated and refined focus on •Improving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or in vitro plantlets.•Obtaining nuclei suspensions through either lab homogenizers or by chopping.•Increasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488 nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. The obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies.

In Situ Gelling Liquid Crystalline System as Local siRNA Delivery System.

An effective short interfering RNA (siRNA) delivery system protects the siRNA from degradation, facilitates its cellular uptake, and promotes its release into the cytoplasm. Local administration of siRNA presents advantages over systemic administration, such as the possibility to use lower doses and allow local and sustained release. In this context, in situ solidifying organogels based on monoglycerides (MO), polyethylenimine (PEI), propylene glycol (PG) and tris buffer are an attractive strategy for intratumoral delivery of siRNA. In this study, precursor fluid formulation (PFF) composed of MO/PEI/PG/tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) was used to deliver siRNA to tumor cells. The internal structure of the gel obtained from PFF was characterized using small angle X-ray scattering (SAXS). In addition, its ability to complex siRNA, protect it from degradation, and functionally deliver it to tumor cells was investigated. Moreover, in vivo gel formation following intratumoral injection was evaluated. The gel formed in excess water from PFF was found to comprise a mixture of hexagonal and cubic phases. The system was able to complex high amounts of siRNA, protect it from degradation, promote siRNA internalization, and induce gene silencing in vitro in a variety of tumor cell lines. Moreover, a gel formed in situ following intratumoral injection in a murine xenograft model. In conclusion, PFF is a potential delivery system for local and sustained delivery of siRNA to tumor tissue after intratumoral administration.

An in situ gelling liquid crystalline system based on monoglycerides and polyethylenimine for local delivery of siRNAs.

The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC2) in contact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC2 formed and was crucial for complexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10 µM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7 days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the formulations, and the gels were degraded in 30 days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14 days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA.

Altered heart rate and blood pressure variability in mice lacking the Mas protooncogene.

Heart rate variability is a relevant predictor of cardiovascular risk in humans. A significant genetic influence on heart rate variability is suggested, although the genes involved are ill-defined. The Mas-protooncogene encodes a G-protein-coupled receptor with seven transmembrane domains highly expressed in testis and brain. Since this receptor is supposed to interact with the signaling of angiotensin II, which is an important regulator of cardiovascular homeostasis, heart rate and blood pressure were analyzed in Mas-deficient mice. Using a femoral catheter the blood pressure of mice was measured for a period of 30 min and 250 data values per second were recorded. The mean values and range of heart rate and blood pressure were then calculated. Neither heart rate nor blood pressure were significantly different between knockout mice and controls. However, high resolution recording of these parameters and analysis of the data by non-linear dynamics revealed significant alterations in cardiovascular variability in Mas-deficient animals. In particular, females showed a strong reduction of heart rate variability. Furthermore, the data showed an increased sympathetic tone in knockout animals of both genders. The marked alterations detected in Mas-deficient mice of both genders suggest that the Mas-protooncogene is an important determinant of heart rate and blood pressure variability.

Experiences from corrosion control of soft and acidic waters

Se analiza la corrosión en las cañerías de distribución de agua tanto exterior como interior que producen altos costos de mantenimiento, siendo el efecto más importante el deterioro de la calidad de agua. Hay muchos procesos de corrosión desconocidos y descontrolados. Se estudia este tema en detalle y los métodos de tratamiento de agua para contrarrestar la corrosión. Se indican métodos de control y se analizan dos casos reales