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Introduction to Cells, DNA, and Viruses
Published in Patricia G. Melloy, Viruses and Society, 2023
Finally, virologists who observe viral particles in tissues might turn to microscopy techniques such as electron microscopy and fluorescence microscopy to study viral protein structure and their interaction with cells. Because of the high magnification and resolution capability of electron microscopy, this microscopy technique has historically been used most often to visualize viruses (Figure 1.5) (Alberts et al. 2019; Lostroh 2019). The techniques of atomic force microscopy and a specialized type of electron microscopy called cryo-electron microscopy are often used as well. Superresolution microscopy has increased the capabilities of light microscopy as well (Nobel Prize Outreach AB 2014). However, cellular changes resulting from viral infection can be observed using conventional light microscopy and have been used even before the viral particles themselves could be visualized (Lostroh 2019).
Roundabouts
Published in John R. Helliwell, The Whats of a Scientific Life, 2019
Are there other options rather than repeat the whole circle of experiments again? One can try a method altogether different than X-ray crystal structure analysis. A method that has improved a lot in the last ten years or so in my field of structural chemistry and biology is that of cryo electron microscopy. In this case, a crystal is not needed and instead multiple images of a single molecular complex are studied directly. This new method has allowed the breaking out of going round in circles for those impossible-to-crystallise cases. Why were they impossible to crystallise? This could be for a variety of reasons. Firstly, although a homogenously pure sample of these molecules could be prepared, they can each be too flexible to crystallise in a simple, repeating, 3D array. Secondly, it may not have been possible to prepare a pure enough sample for crystallisation. So important is the improved method of cryo electron microscopy for the field of structural biochemistry and structural biology that its lead developers shared the Nobel Prize for Chemistry in 2017: Jacques Dubochet, Joachim Frank and Richard Henderson [2].
Liposomes
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
Size distribution is normally measured by dynamic light scattering. This method is reliable for liposomes with relatively homogeneous size distribution, while various bimodal size distributions may more reflect mathematical algorithms used in calculation to fit a multiexponential decay function than reflect the properties of the sample. A very simple but very powerful method is gel exclusion chromatography where truly hydrodynamic radius can be detected if the column is well calibrated. The line width of the peak indicates size distribution. For liposomes only Sephacryl S 1000 has pores large enough to separate liposomes. This column (typically 30 cm, diameter 1 cm, flow rate 5 to 8 mL/h, applied 1 to 5 μM of liposomes) can separate liposomes in the size range 30 to 300 nm. Sepharose 4B and 2B columns can separate SUV from micelles. All these columns are more difficult to operate with positively charged colloidal particles because of possible electrostatic interactions with gel medium, which can be slightly negatively charged, and because the addition of salt can cause the aggregation of the sample and clogging of the column. Many researchers use electron microscopy to measure liposome size. The most widely used negative staining and freeze-fracture methods are prone to artifacts due to the changes during sample preparation as well as to geometric reasons: it is difficult to estimate the size of collapsed dry vesicles in the negative stain procedure while in freeze-fracture or thin sectioning it is not known in which plane vesicles were fractured or cut. A novel development — cryoelectron microscopy — where a sample is frozen and directly observed in the electron beam, without any staining, shadowing, or replica preparation, is much more reliable (see Figure 6-9).
Electron microscopy overview of SARS-COV2 and its clinical impact
Published in Ultrastructural Pathology, 2022
Soheir Saiid Mansy, Mona Mahmoud AbouSamra
Many techniques, including NMR spectroscopy, X-ray solution scattering, neutron diffraction, various spectroscopic techniques, and X-ray crystallography, have been used to determine the shape and structure of biological molecules. Recently, cryo-electron microscopy has become the most effective tool in structural biology after the technical development of its resolutions, which permits the identification of the biomolecular structure in its natural state.59 Cryo-EM has an advantage over X-ray crystallography, and is the most effective tool in analyzing macromolecules during the last few years. Cryo-EM reveals structures in fast-frozen non-crystalline biological samples that are closer to their natural state at an atomic level. In addition, it requires much smaller macromolecule samples to work with, unlike X-ray crystallography, which needs large pieces of materials to optimize the crystallization conditions.59 Hence, cryo-EM has become the tool of choice for determining the structure of macromolecular complexes, especially supra-assemblies that are difficult to prepare in large quantities or virtually inaccessible to crystallize.59,61,62 Identifying the structural biology of viral protein complexes at molecular resolution is important for designing small drug molecules to bind and impair their function.32
A beacon for broader impact: illuminating science
Published in Journal of Visual Communication in Medicine, 2019
Our time is one of rapid rate of change, especially in science and technology; it is known as the ‘The Digital Age’, ‘The Century of Biology’, and ‘The Information Age’. With new advances in imaging, like cryo-electron microscopy, we have insight into biological structures like never before. With technologies in science, such as CRISPR-Cas9 genome editing, a system adept at knocking out mutations, researchers have opened a new gamut of potential applications in genetics.
Glutaminase inhibitors: a patent review
Published in Expert Opinion on Therapeutic Patents, 2018
CanRong Wu, LiXia Chen, Sanshan Jin, Hua Li
Moreover, the adjustment and modification of the compound skeleton based on the same regulatory site can only optimize its inhibition potency and pharmacokinetics. For instance, a large number of inhibitors derived from the structure of BPTES have been disclosed in the past five years. Although these novel inhibitors could be unique in their GLS inhibitory potency, the on-target activity, pharmacokinetic and physiochemical properties, their modes of action are unlikely to be distinguished from clinical candidate CB-839. The discovery of allosteric inhibitor with other mechanisms therefore remains very meaningful. For example, compound 968, which was screened from small compound library in Cornell University, exhibited different allosteric inhibition mechanism from BPTES. Due to the lack of co-crystal structure of compound 968 and GAC, structural optimization of the compound is greatly restricted. Although dozens of derivatives of compound 968 were synthesized by researchers in Cornell University, they only obtained a very limited optimization, with the IC50 value reduced only by five times. Therefore, it is important to obtain the structure of the complex to guide the further structural optimization. Perhaps due to its unique allosteric regulation mechanism, it is very difficult to obtain the co-crystal structure of compound 968 and GAC or KGA. Technological advances in cryo-electron microscopy have made it possible to be used in drug discovery [108]. And the molecular weight of tetramers GAC has reached 230 KD, which is suitable for using cryo-electron microscopy to resolve the complex structure. The detailed ligand-receptor interactions information from their complex structure not only guides the rational drug design, but also facilitates in silico structure-based virtual ligand screening of novel GLS inhibitors, which makes the discovery of novel chemical scaffolds more economical and efficient. For instance, based the co-crystal structure of KGA and BPTES (PDB code: 3VP1), Wu CR et al. [64] found that a new natural product by virtual ligand screening method could reversibly inhibit KGA activity and showed excellent antitumor activity both in vitro and in vivo.