Cutting Edge Techniques in Biophysics, Biochemistry and Cell Biology: From Principle to Applications

Animal cell culture techniques in today’s scenario have become an indispensable tool in the field of biomedical research. It provides a basis to study molecular and biochemical changes associated with disease pathogenesis. It explicitly provides a scope to study gene expressions, regulation, proliferation, and differentiation in normal as well as pathologic conditions. The culturing of animal cells requires aseptic conditions and vital technical skills to carry out successful cell culture experiments. It provides an appropriate model for studying cell and molecular biology, biochemical changes in cells, drug screening and efficacy etc. This chapter describes the essential techniques of animal cell culture as well as its applications.

The present chapter highlights the various newer and advanced real-time quantitative PCR (qPCR) detection chemistries and their applications in biomedical research and molecular diagnostics. The chapter also summarizes the most advanced modification of the qPCR based technique along with next generation methods which have immense potential to revolutionize the field of biomedical sciences and molecular diagnostics.

Flow cytometer is a sophisticated and analytical tool which analyzes the phenotypic and functional characteristics of a cell in much lesser time. It measures the light scattering features, mainly forward scatter and side scatter properties of the cell. Optics, fluidics, and electronics are the main elements flow cytometer. Hydrodynamic focusing of the sheath fluid present in a sheath chamber enables the streamline motion of cells. This allows the encounter of each cell separately with the laser. A photodiode, photomultiplier tubes, optical filters, and beam splitters collect and emit the light or fluorescence in the form of signals. The signals are further processed by the electronic component and with the help of software, data are analyzed. For a cell to emit fluorescence, a number of fluorochromes (FITC, Alexa Fluor 488, Alexa Fluor 647 etc.) and dyes (propidium iodide, Hoechst, ethidium bromide etc.) are available. These fluorochromes may be conjugated with antibodies and emit fluorescence when excited by the laser. A diverse range of applications are possible using flow cytometer like apoptosis assay, cell cycle analysis, free radical generation assay, cytokine estimation, immunophenotyping, phagocytosis assay and many more. An advanced application of flow cytometer receiving attention is cell sorting. Individual cells or cell types can be sorted from a mixture of cells using physical fluorescence emission features of the cells named fluorescence assisted cell sorter.

In X-ray crystallography, the phase problem is a major bottle neck, it would be successful only if we obtain a suitable template or phases can be determined experimentally. However molecular replacement calculation is the most useful technique and it could be applied when there is a homologous structure known. To obtain the phase information, the most widely used method is derivatization of heavyatoms for protein crystals, although it is generally not used nowadays. While it is a valuable technique to obtain the phases for unidentified structures having no identity with the homologous for using molecular replacement (MR) and the crystals that cannot be diffracted even at synchrotron, nowadays, the SAD and MAD methods for experimental phasing have been developed. The more advanced technique has also been developed that is Cryo-EM and SFX with XFELs, having enormous potential for determining the structure of novel proteins that are not acquiescent to produce a crystal that cannot diffract.

With the increasing demand for commercially important proteins, it has become essential to develop more improved and efficient methods for heterologous protein production to meet the requirements of society. Although currently there are different host systems available for the production of proteins heterologously, still the selection of a perfect host system becomes a tedious task if the aim is to get fully functional protein in adequate quantity. Different types of hosts are commonly used for heterologous protein production include Escherichia coli, Saccharomyces cerevisiae, insect cell expression systems and various mammalian systems. Different host systems that are used for heterologous protein expression have their own advantages and disadvantages as in the case of bacterial systems, a good amount of protein is produced but the quality of the protein may not be appropriate. Similarly, eukaryotic hosts also suffer from specific merits and demerits. Different promoter systems that are used for gene expression also influence protein production in case of both prokaryotic and eukaryotic hosts. The selection of the appropriate host depends on the amount of the protein, quality of the protein in terms of functional activity and its application. In this chapter, we will discuss the principle of heterologous gene expression, different types of hosts organism available for heterologous protein production. We will also present the current developments and modifications which are being done in various host systems to improve the process of protein production. We will elaborate on the different types of vectors that are used for recombinant protein production, their contrasting features and different promoter systems used in the vectors, importance or promoters with respect to protein production. In addition to this, we will also elaborate on different aspects related to the overexpression and purification of recombinant heterologous protein and the type of tags and chromatographic techniques used for the purification of different types of proteins.

Rational drug designing encompasses several theoretical methods and in silico approaches involving molecular modeling, docking etc. to study the behavior and the properties of molecular systems. Specifically, the techniques employed in the fields of computational chemistry, computational biology, nanotechnology, and material science vary in complexity, and theoretical observations depend on the system type and the system size being investigated. Molecular modeling involves both quantum and molecular mechanics (QM & MM). The fundamental concepts of molecular modelling with suitable examples are elaborated. The present chapter also highlights the sequential flow of in silico approaches used for the purpose of drug designing, giving a brief snapshot of the software used for this purpose. The advanced techniques and applications of computational methods used for new drug development are explained. Thus, this chapter provides an insight into the various dimensions of computer-aided drug design; its driving force, recent development, and future prospects.

X-ray crystallography has immensely contributed to the growth of the science of understanding the three-dimensional structure of matters. The atomic arrangement of small molecules such as salts, inorganic, organic complexes, and metallic compounds was determined. Later on, one after another flood of molecular structures from biological origins was solved using X-ray crystallography. The structure of DNA was determined using fiber diffraction methods in the 1950s, subsequently, structures of polysaccharides, fibrous proteins, and virion particles were determined. The crystal structures of the first protein molecules in the form of lysozyme, myoglobin, and hemoglobin were the enormous achievements of the 1960s, solved by single-crystal diffraction methods. Within a couple of decades later, atomic structures of viruses and membrane receptors were started to be determined. Currently, there are over 125 thousand crystal structures submitted to the PDB database at the rate of more than 3 thousand structures per year. In contrast, there are 12 thousand structures solved by NMR spectroscopy at the rate of just over 100 structures per year, whereas there are only 2 thousand structures available in PDB which are solved using computational methods. It shows the popularity of X-ray crystallography for revealing the atomic details of protein molecules in the field of structural biology. For determining the structure, the molecule is first crystallized to have a repetitive and regular arrangement of arrays in three-dimensional space. As the X-rays have a wavelength in the order of bond distances existing in matters, they are the suitable electromagnetic radiations to be used for finding detailed atomic positions. A beam of X-rays is diffracted from the crystalline matter and is collected at certain positions. The intensities, amplitude, and phases of the diffracted X-rays are convoluted to calculate the electron density of atoms in the crystal. The atomic positions are refined by putting them at mean positions in the electron density and eventually the atomic coordinates in 3-D space are revealed, which define the shape of the matter or a molecule. Biomolecular crystallography deals with the crystal structure determination of biomolecules such as proteins, nucleic acids, polysaccharides, complexes, etc. As the structure and the function of a biomolecule are closely associated, revealing the structure is incredibly advantageous in order to understand or alter the function of the biomolecules. This understanding has given rise to the advent of structure-based drug discovery methods. The available 3-D structure of a druggable target protein may also be used for structure-based drug design against a pathophysiological state.

In the last two decades, the development of massive parallel sequencing methods has allowed the sequencing of RNA at an unprecedented resolution, unleashing an enormous wealth of information about the cellular state. Sequencing has accelerated biomedical research by identifying novel mutations, aberrant splicing patterns, splicing isoforms, new gene regulators, and cell-to-cell heterogeneity. In order to efficiently characterize the complexity of the complete transcriptome, there is a steady development for different RNA sequencing [RNA-seq] protocols by improving different steps from library preparation to the data analysis. Furthermore, with the advancements in the sequencing strategies, single-cell RNA sequencing[scRNA-seq] methods have been developed allowing to address the heterogeneity in cell types, and mRNA expression at a remarkable resolution. The majority of these methods involve the conversion of RNA to cDNA and thus amenable to errors, PCR and ligation biases, and inefficiencies of enzymes. Amid these challenges, strategies have been developed to sequence the RNA directly at the single-molecule level which allows to overcome these biases. This chapter provides a brief overview of different sequencing technologies available for the RNA-seq, scRNA-seq and single molecule RNA sequencing along with the different aspects where RNA sequencing has contributed to the biomedical field.

Antigens and immunoglobulins are important biological entities. Interaction of these components is being considered as a major criterion to assess the health status in veterinary and medical science. Antigen-antibody reactions are highly specific. This interaction between antigen and antibody has been used as the principle of immunoelectrophoresis. This technique has got tremendous applications in basic and applied health science. The present chapter describes principle, process, types, applications, advantages, and limitations of various types of immunoelectrophoresis. This information will be very useful for the students of immunology and scholars who need to develop an understanding of immunoelectrophoresis and its applications.