Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.
Nanoparticles have recently gained increased attention as drug delivery systems for the treatment of cancer due to their minute size and unique chemical properties. However, very few studies have tested the biophysical changes associated with nanoparticles on metastatic cancer cells at the cellular and sub-cellular scales. Here, we investigated the mechanical and morphological properties of cancer cells by measuring the changes in cell Young's Modulus using AFM, filopodial retraction (FR) by time lapse optical light microscopy imaging and filopodial disorganization by high resolution AFM imaging of cells upon treatment with nanoparticles. In the current study, nanomechanical changes in live murine metastatic breast cancer cells (4T1) post exposure to a nanodiamond/nanoplatinum mixture dispersed in aqueous solution (DPV576), were monitored. Results showed a decrease in Young's modulus at two hours post treatment with DPV576 in a dose dependent manner. Partial FR at 20 min and complete FR at 40 min were observed. Moreover, analysis of the retraction distance (in microns) measured over time (minutes), showed that a DPV576 concentration of 15%v/v yielded the highest FR rate. In addition, DPV576 treated cells showed early signs of filopodial disorganization and disintegration. This study demonstrates the changes in cell stiffness and tracks early structural alterations of metastatic breast cancer cells post treatment with DPV576, which may have important implications in the role of nanodiamond/nanoplatinum based cancer cell therapy and sensitization to chemotherapy drugs.
Biological model of hypoxia can be used for the diagnosis o f functional changes in human erythrocytes under the effect of the hypoxic factor. The use of this model together with mod ern methods of scanning probe microscopy for evaluation o f the severity of pulmonological disease in senile patients will help to predict treatment efficiency and outcome. Key W ords: scanning probe microscopy (SPM); Young' s modulus; pneumoniaPhysiological changes in human tissues during aging manifest in impaired adaptation capacity to metabolic stress and general deterioration of the health status. The older the individual, the higher is the risk o f disease development, o f which pulmonological diseases, in cluding acute community-acquired pneumonia (called pneumonia in the text below) are most hazardous [1]. Respiratory diseases often cause a potent unfavorable effect, oxygen deficit in tissues, and therefore, studies of tissue hypoxia is the main problem o f pulmonol ogy and physiology [2]. Studies o f the mechanisms of cell response in hypoxia, evaluation of regularities of disease development, and the search for prognostic and diagnostic criteria are expected to improve evalu ation of the severity and progress o f various diseases, including pneumonia [8]. Scanning probe microscopy (SPM) is an informative method for studies o f the physiological status in health and disease, specifically, for studies o f blood cell parameters. Extrapolation o f experimental data on animal blood obtained on a biological model to the data ob tained in studies of hypoxia in patients is expected to show the trend of changes in blood cell structure and functions, indicating the effect of the hypoxic stress factor on the organism.The strong impact of infection is essential in pneu monia, which can modulate the cell parameters, specif ically the erythrocyte values [1]. Use o f the biological model will help differentiate the hypoxic effects on changes in human blood erythrocyte characteristics from the bacteriological factor. If the trend of changes in the rat red blood cells is similar to changes in pa tients with pneumonia, presumably, it is the hypoxic, but not the bacterial factor, that is mainly responsible for changes in human erythrocytes.We used the biological model for SPM studies of the blood cell structure and functions in senile patients with pneumonia.
We propose and tested a method for studies of native blood cells by atomic-force microscopy in a humid chamber preserving viability, size, and shape of biological objects. The method has some advantages over scanning in a liquid cell: it allows studying non-fixed blood samples in the form of suspension of live cells and excludes mechanical and chemical influences on the cells.
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