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Host Defenses Against Prototypical Intracellular Protozoans, the Leishmania
Published in Peter D. Walzer, Robert M. Genta, Parasitic Infections in the Compromised Host, 2020
Richard D. Pearson, Mary E. Wilson
Although minor ultrastructural differences exist, it is impossible to reliably differentiate between Leishmania species on the basis of promastigote or amastigote morphology (8-14). Consequently, the original classification of species was based on the clinical syndrome produced in humans, epidemiological differences, geographical distribution, involvement of specific animal reservoirs, and transmission by different sandfly species. Several biochemical methods subsequently became available: isoenzyme pattern (15-19), buoyant density analysis of nuclear or kinetoplast DNA (16,20), radiorespirometry (21), and antigenic differences in promastigotes or their excreted factors (22,23), Isoenzyme analysis has been one of the most widely used. Species-specific monoclonal antibodies (24) and restriction endonucelase analysis or hybridization of kinetoplast DNA (24-27) have recently emerged as effective methods of speciation. Although there is no consensus as to the best method for speciation, there has been general agreement among the various biochemical methods. Recently, pulsed field gradient gel electrophoresis has been used to fractionate chromosomes of Leishmania species (28), and the karyotypes of species have been found to differ (29).
Molecular Organization of Entamoeba Histolytica
Published in Roberto R. Kretschmer, Amebiasis: Infection and Disease by Entamoeba histolytica, 2020
Isaura Meza, Haydee K. Torres-Guerrero, Marco A. Meraz
Total content of DNA in E. histolytica trophozoites estimated by several methods indicates great variability.92,93 Recent estimates are 0.5 pg/nucleus for E. histolytica HMI and a genome size of 4.0 × 108. All of the DNA is in the nucleus as there are no mitochondria or other DNA-containing organelles. Sizing of the DNA, by pulse field gradient electrophoresis, indicates at least seven fragments from 50 to 500 kb.7 Nothing is known about the ploidy in Entamoeba. The possibility of having amplification of rDNA10,92 as well as having modified bases could explain the differences in DNA and GC content reported in the literature. The latter ranges from 27 to 38% for E. histolytica.7,92,94
Fingerprinting Techniques for Herbal Drugs Standardization
Published in Ravindra Kumar Pandey, Shiv Shankar Shukla, Amber Vyas, Vishal Jain, Parag Jain, Shailendra Saraf, Fingerprinting Analysis and Quality Control Methods of Herbal Medicines, 2018
Ravindra Kumar Pandey, Shiv Shankar Shukla, Amber Vyas, Vishal Jain, Parag Jain, Shailendra Saraf
The combination of liquid chromatography (LC) and nuclear magnetic resonance (NMR) offers the potential of unparalleled chemical information from analytes separated from complex mixtures. This separation technique with NMR spectroscopy is one of the most powerful and time saving methods for the separation and structural elucidation of unknown compounds and mixtures, especially for the structural elucidation of light and oxygen sensitive substances. The online LC-NMR technique allows the continuous registration of time changes as they appear in the chromatographic run automated data acquisition and processing in LC-NMR improves the speed and sensitivity of the detection. The recent introduction of a pulsed field gradient technique in high resolution NMR as well as a three-dimensional technique improves the application for structure elucidation and molecular weight information. These new hyphenated techniques are useful in the areas of pharmacokinetics, toxicity studies, drug metabolism, and drug discovery processes. Several other hyphenated NMR techniques have been developed to enhance the sensitivity of this technique. Liquid chromatography-solid-phase extraction-nuclear magnetic resonance (LC-SPE-NMR) increases the sensitivity of the instrument by utilizing a solid phase extraction device after the LC column. Capillary LC-NMR also practically lowers the detection limit to a nanogram range through the integration of capillary LC with NMR detection (Walker and O'Connell, 2008; Wang et al., 2001).
Updated insight into the characterization of nano-emulsions
Published in Expert Opinion on Drug Delivery, 2023
Xinyue Wang, Halina Anton, Thierry Vandamme, Nicolas Anton
Besides its common use for chemical characterization, in recent years, NMR was also used in NE characterization. This method reveals information on the arrangement of surfactants and co-surfactant on the oil-water interface by the modification of their chemical shift. Xie et al. chose 1H and 13C NMR as the major approach to identify the functional groups on the droplet’s interface, through following the variation of the peak intensity and comparing formulations with different interfacial composition [92]. Another NMR-based technology, so-called pulsed field gradient NMR (PFG-NMR), can be used to determine the particle size and size distribution for single and double emulsions [20]. This technique analyzes the reduction of the molecular diffusion, when they are confined in the discrete phase – i.e. emulsion droplets – in comparison to those located in the interfacial region. Analyzing when the diffusion becomes restricted provides an estimation of the droplet size. Beyond the information in size, PFG-NMR allows the fine characterization of assembled states and structures, level of sedimentation, or creaming of emulsions [93]. Figure 10 illustrates the size determination of the droplet suspension by PFG-NMR, showing that this technique is less affected by light scattering and permits to track the diffusion properties of individual components in turbid and concentrated samples, by measuring their characteristic spectra (inherently chemically selective).
Capsules from synthetic diblock-peptides as potential artificial oxygen carriers
Published in Journal of Microencapsulation, 2021
Huayang Feng, Jürgen Linders, Sascha Myszkowska, Christian Mayer
This study is meant to explore the feasibility of synthesising amphiphilic diblock-peptides by a phosgene-free method and their potential application for PFD-filled capsules (Scheme 1). The polypeptide consists of blocks of poly aspartic acid and poly phenylalanine, whose chemical structure is identified using 1H-NMR. The morphology and size distribution of the PFD-filled capsules is investigated with atomic force microscopy (AFM) and particle tracking via video microscopy (VM). The molecule diffusion and gas exchange properties of the capsules are studied by Pulsed Field-Gradient Nuclear Magnetic Resonance (PFG-NMR) and 19F-NMR. The results indicate that diblock-peptides synthesised by the proposed phosgene-free method lead to PFD-filled capsules which are very suitable as artificial oxygen carriers. Of course, in order to be applicable as oxygen carriers for artificial blood replacement, the capsules will have to pass further testing, e.g. for blood compatibility, cellular toxicity, allergenic potential, and metabolic degradation. But being composed only of canonical amino acids and offering a wide variety towards their choice, chances are very good to reach this target in future developments.
Quantification of natural abundance NMR data differentiates the solution behavior of monoclonal antibodies and their fragments
Published in mAbs, 2021
David Ban, Cory T. Rice, Mark A. McCoy
The test set of Ab(L/F)s molecules was probed by DOSY/Diffusion NMR following previously published protocols.34 Diffusion NMR was used to determine the translational diffusion coefficient (Dt) for a given molecule. Diffusion NMR experiments were conducted using conventional pseudo 2D Pulse Field Gradient (PFG) filtered 1Ds where the gradient amplitude was varied from 5% to 95% of the maximum value tolerated by the probe and a total of 32 different gradient amplitudes were collected per sample. The delay between recovery gradients was set to 300 ms where each gradient was applied for 1.5 ms. The maximum gradient amplitude was set to 58.9 G⋅cm−1. Each 1D was adopized using a shifted sine-bell and zero-filled prior to Fourier transform.