Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing

Chitosan, a well known natural cationic polysaccharide, has been successfully implemented in vitro and in vivo as a nonviral delivery system for both plasmid DNA and siRNA. While using chitosan/siRNA polyplexes to knock down specific targets, we have underestimated the effect of nucleic acids binding to chitosan when extracting RNA for subsequent quantitative PCR evaluation of silencing. In vitro transfection using chitosan/siRNA-based polyplexes reveals a very poor recovery of total RNA especially when using low cell numbers in 96 well plates. Here, we describe a method that dramatically enhances RNA extraction from chitosan/siRNA-treated cells by using an enzymatic treatment with a type III chitosanase. We show that chitosanase treatment prior to RNA extraction greatly enhances the yield and the integrity of extracted RNA. This method will therefore eliminate the bias associated with lower RNA yield and integrity when quantifying gene silencing of chitosan-based systems using quantitative real time PCR

Spectral averaging techniques for Jacobi matrices

Spectral averaging techniques for one-dimensional discrete Schroedinger operators are revisited and extended. In particular, simultaneous averaging over several parameters is discussed. Special focus is put on proving lower bounds on the density of the averaged spectral measures. These Wegner type estimates are used to analyze stability properties for the spectral types of Jacobi matrices under local perturbations

Low molecular weight chitosan nanoparticulate system at low N:P ratio for nontoxic polynucleotide delivery

Chitosan, a natural polymer, is a promising system for the therapeutic delivery of both plasmid DNA and synthetic small interfering RNA. Reports attempting to identify the optimal parameters of chitosan for synthetic small interfering RNA delivery were inconclusive with high molecular weight at high amine-to-phosphate (N:P) ratios apparently required for efficient transfection. Here we show, for the first time, that low molecular weight chitosan (LMW-CS) formulations at low N:P ratios are suitable for the in vitro delivery of small interfering RNA. LMW-CS nanoparticles at low N:P ratios were positively charged (ζ-potential ~20 mV) with an average size below 100 nm as demonstrated by dynamic light scattering and environmental scanning electron microscopy, respectively. Nanoparticles were spherical, a shape promoting decreased cytotoxicity and enhanced cellular uptake. Nanoparticle stability was effective for at least 20 hours at N:P ratios above two in a slightly acidic pH of 6.5. At a higher basic pH of 8, these nanoparticles were unravelled due to chitosan neutralization, exposing their polynucleotide cargo. Cellular uptake ranged from 50% to 95% in six different cell lines as measured by cytometry. Increasing chitosan molecular weight improved nanoparticle stability as well as the ability of nanoparticles to protect the oligonucleotide cargo from nucleases at supraphysiological concentrations. The highest knockdown efficiency was obtained with the specific formulation 92-10-5 that combines sufficient nuclease protection with effective intracellular release. This system attained >70% knockdown of the messenger RNA, similar to commercially available lipoplexes, without apparent cytotoxicity. Contrary to previous reports, our data demonstrate that LMW-CS at low N:P ratios are efficient and nontoxic polynucleotide delivery systems capable of transfecting a plethora of cell lines

Seabed images from Southern Ocean shelf regions off the northern Antarctic Peninsula and in the southeastern Weddell Sea

Recent advances in underwater imaging technology allow for the gathering of invaluable scientific information on seafloor ecosystems, such as direct in situ views of seabed habitats and quantitative data on the composition, diversity, abundance, and distribution of epibenthic fauna. The imaging approach has been extensively used within the research project DynAMo (Dynamics of Antarctic Marine Shelf Ecosystems) at the Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research Bremerhaven (AWI), which aimed to comparatively assess the pace and quality of the dynamics of Southern Ocean benthos. Within this framework, epibenthic spatial distribution patterns have been comparatively investigated in two regions in the Atlantic sector of the Southern Ocean: the shelf areas off the northern tip of the Antarctic Peninsula, representing a region with above-average warming of surface waters and sea-ice reduction, and the shelves of the eastern Weddell Sea as an example of a stable high-Antarctic marine environment that is not (yet) affected by climate change. The AWI Ocean Floor Observation System (OFOS) was used to collect seabed imagery during two cruises of the German research vessel Polarstern, ANT-XXIX/3 (PS81) to the Antarctic Peninsula from January to March 2013 and ANT-XXXI/2 (PS96) to the Weddell Sea from December 2015 to February 2016. Here, we report on the image and data collections gathered during these cruises. During PS81, OFOS was successfully deployed at a total of 31 stations at water depths between 29 and 784 m. At most stations, series of 500 to 530 pictures ( >  15 000 in total, each depicting a seabed area of approximately 3.45 m2 or 2.3  ×  1.5 m) were taken along transects approximately 3.7 km in length. During PS96, OFOS was used at a total of 13 stations at water depths between 200 and 754 m, yielding series of 110 to 293 photos (2670 in total) along transects 0.9 to 2.6 km in length. All seabed images taken during the two cruises, including metadata, are available from the data publisher PANGAEA via the two persistent identifiers at https://doi.org/10.1594/PANGAEA.872719 (for PS81) and https://doi.org/10.1594/PANGAEA.862097 (for PS96)

Compellingly high SARS-CoV-2 susceptibility of Golden Syrian hamsters suggests multiple zoonotic infections of pet hamsters during the COVID-19 pandemic

Golden Syrian hamsters (Mesocricetus auratus) are used as a research model for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Millions of Golden Syrian hamsters are also kept as pets in close contact to humans. To determine the minimum infective dose (MID) for assessing the zoonotic transmission risk, and to define the optimal infection dose for experimental studies, we orotracheally inoculated hamsters with SARS-CoV-2 doses from 1 * 105 to 1 * 10-4 tissue culture infectious dose 50 (TCID50). Body weight and virus shedding were monitored daily. 1 * 10-3 TCID50 was defined as the MID, and this was still sufficient to induce virus shedding at levels up to 102.75 TCID50/ml, equaling the estimated MID for humans. Virological and histological data revealed 1 * 102 TCID50 as the optimal dose for experimental infections. This compelling high susceptibility leading to productive infections in Golden Syrian hamsters must be considered as a potential source of SARS-CoV-2 infection for humans that come into close contact with pet hamsters

Potassium binding adjacent to cationic transition metal fragments: unusual heterobimetallic adducts of a calix[4]arene-based thione ligand

The synthesis of cationic rhodium and iridium complexes of a bis(imidazol-2-thione) functionalised calix[4]arene ligand and their surprising capacity for potassium binding is described. In both cases uptake of the alkali metal into the calix[4]arene cavity occurs despite adverse electrostatic interactions associated with close proximity to the transition metal fragment (Rh+∙∙∙K+ = 3.715(1) Å, Ir+∙∙∙K+ = 3.690(1) Å). The formation and constituent bonding of these unusual heterobimetallic adducts has been interrogated through extensive solution and solid-state characterisation, examination of the host-guest chemistry of the ligand and its upper-rim unfunctionalised calix[4]arene analogue, and computationally using DFT-based energy decomposition analysis (EDA)

Understanding the Random Displacement Model: From Ground-State Properties to Localization

We give a detailed survey of results obtained in the most recent half decade which led to a deeper understanding of the random displacement model, a model of a random Schr\"odinger operator which describes the quantum mechanics of an electron in a structurally disordered medium. These results started by identifying configurations which characterize minimal energy, then led to Lifshitz tail bounds on the integrated density of states as well as a Wegner estimate near the spectral minimum, which ultimately resulted in a proof of spectral and dynamical localization at low energy for the multi-dimensional random displacement model.Comment: 31 pages, 7 figures, final version, to appear in Proceedings of "Spectral Days 2010", Santiago, Chile, September 20-24, 201