Alzheimer's disease (AD) affects millions of people world-wide and new effective and safe therapies are needed. Cotinine, the main metabolite of nicotine, has a long half-life and does not have cardiovascular or addictive side effects in humans. We studied the effect of cotinine on amyloid-β (Aβ) aggregation as well as addressed its impact on working and reference memories. Cotinine reduced Aβ deposition, improved working and reference memories, and inhibited Aβ oligomerization in the brains of transgenic (Tg) 6799 AD mice. In vitro studies confirmed the inhibitory effect of cotinine on Aβ1-42 aggregation. Cotinine stimulated Akt signaling, including the inhibition of glycogen synthase kinase 3β (GSK3β), which promotes neuronal survival and the synaptic plasticity processes underlying learning and memory in the hippocampus and cortex of wild type and Tg6799 AD mice. Simulation of the cotinine-Aβ1-42 complex using molecular dynamics showed that cotinine may interact with key histidine residues of Aβ1-42, altering its structure and inhibiting its aggregation. The good safety profile in humans and its beneficial effects suggest that cotinine may be an excellent therapeutic candidate for the treatment of AD.
Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. L-lysine and L-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used L-lysine and L-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations.
a-Synuclein, a natively unstructured protein important in the neuropathology of Parkinson’s disease, was found to form a Langmuir monolayer in an a-helical conformation with its helical axis parallel to the air–water interface. This study sheds light on the role of vesicles in neuronal cells in the accumulation/aggregation of a-synuclein.
Control of column loading in Protein A chromatography is a crucial part of development of robust and flexible process platforms for continuous production of monoclonal antibody (mAb) products. In this paper, we propose a control system that uses near infrared spectroscopy (NIRS) flow cells to accomplish the above. Two applications have been demonstrated using a periodic counter-current continuous chromatography setup. The first application involves use of single NIR flow cell before the inlet of the loading column to measure the concentration of mAb in the harvested broth. Measurement was in real-time (every 3 s) and within ±0.05 mg/ml, significantly better than making UV-based concentration estimations. The second application involved use of an additional NIR flow cell at the outlet of the loading column to measure column breakthrough in real time. The concentration data was transferred to a Python-based monitoring and control algorithm layered over a Cadence BioSMB system. The program could successfully run a three-column periodic counter current method on the BioSMB whereas controlling loading to ensure optimal resin utilization in each loading cycle phase based on precharacterized dynamic binding capacity models, whereas maintaining periodic elutions. The system was tested with multiple perturbations in harvest concentration, modeled after deviations that could arise downstream of a perfusion cell culture system. The resultsshow that the proposed control is a spectroscopy-based process analytical technology tool that facilitates real time monitoring and control of loading in process chromatography. It is adaptable to any continuous chromatography equipment and is very well suited for implementation in a continuous mAb production train.
There has been a lack of quick, simple and reliable methods for determination of nanoparticle size. An investigation of the size of hydrophobic (CdSe) and hydrophilic (CdSe/ZnS) quantum dots was performed by using the maximum position of the corresponding fluorescence spectrum. It has been found that fluorescence spectroscopy is a simple and reliable methodology to estimate the size of both quantum dot types. For a given solution, the homogeneity of the size of quantum dots is correlated to the relationship between the fluorescence maximum position (FMP) and the quantum dot size. This methodology can be extended to the other fluorescent nanoparticles. The employment of evolving factor analysis and multivariate curve resolution-alternating least squares for decomposition of the series of quantum dots fluorescence spectra recorded by a specific measuring procedure reveals the number of quantum dot fractions having different diameters. The size of the quantum dots in a particular group is defined by the FMP of the corresponding component in the decomposed spectrum. These results show that a combination of the fluorescence and appropriate statistical method for decomposition of the emission spectra of nanoparticles may be a quick and trusted method for the screening of the inhomogeneity of their solution.
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