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DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine.

DNA comprises molecular information stored via genetic bases (G, C, T, A) and also epigenetic bases, principally 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Both genetic and epigenetic information are vital to our understanding of biology and disease states. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases typically fail to capture common C-to-T mutations or distinguish 5mC from 5hmC. Here, we present a single-base-resolution sequencing methodology that will simultaneously sequence complete genetics and complete epigenetics in a single workflow. The approach is non-destructive to DNA and provides a digital readout of bases, which we exemplify by simultaneous sequencing of G, C, T, A, 5mC and 5hmC; 6-Letter sequencing. We demonstrate sequencing of human genomic DNA and also cell-free DNA taken from a blood sample of a cancer patient. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. We envisage it will be versatile across many applications in life sciences.

Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.

The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

Congenital stationary night blindness (CSNB) is an inherited and non progressive retinal dysfunction. Here we present the crystal structure of CSNB causing T94I(2.61) rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin II state by establishing a direct van der Waals contact with K296(7.43) the site of retinal attachment. This is in stark contrast to the light activated state of the CSNB causing G90D(2.57) mutation where the charged mutation forms a salt bridge with K296(7.43) To find the common denominator between these two functional modifications we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D(2.57) and the hydrophobic T94I(2.61) mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113(3.28) We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.

After 25 years of intensive research, the understanding of how photoreceptors in the eye perceive light and convert it into nerve signals has largely advanced. Central to this is the structural and mechanistic exploration of the G protein-coupled receptor rhodopsin acting as a dim-light sensing pigment in the retina. Investigation of rhodopsin by X-ray crystallographic, electron microscopic, and biochemical means depends on the ability to produce and isolate pure rhodopsin protein. Robust and well-defined protocols permit the production and crystallization of rhodopsin variants to investigate the inactive ground, the fully activated metarhodopsin II state, or disease-causing rhodopsin mutations. This chapter details how we express and purify biologically active variants of rhodopsin from HEK293S GnTI(-) cells in a quality and quantity suitable for biochemical assays, crystallization, and structure determination.

After 25 years of intensive research, the understanding of how photoreceptors in the eye perceive light and convert it into nerve signals has largely advanced. Central to this is the structural and mechanistic exploration of the G protein-coupled receptor rhodopsin acting as a dim-light sensing pigment in the retina. Investigation of rhodopsin by X-ray crystallographic, electron microscopic, and biochemical means depends on the ability to produce and isolate pure rhodopsin protein. Robust and well-defined protocols permit the production and crystallization of rhodopsin variants to investigate the inactive ground, the fully activated metarhodopsin II state, or disease-causing rhodopsin mutations. This chapter details how we express and purify biologically active variants of rhodopsin from HEK293S GnTI(-) cells in a quality and quantity suitable for biochemical assays, crystallization, and structure determination.

All the actions taken and implemented by the Government of a particular country influences the citizens of that country. In November 2016, the Prime Minister of India declared the banknotes of 500 and 1000 rupees as illegal tender. This demonetisation has been called worldwide as the surgical strike on black money as this move aims to reduce corruption and black money in India. However, despite having positive long lasting effects, this decision was met by mixed opinions among the citizens of India. Some lauded this decision while some called it ‘ill-planned’. Although posing some short term inconvenience for the public like limit on withdrawal, long queues outside banks for deposit, withdrawal and exchange of old currency with the new ones, this demonetisation is expected to benefit the economy in the long run. Therefore, it becomes necessary to study what common people think of this demonetisation move. Hence, an attempt has been made in this paper to understand perception of the individuals with regard to demonetisation along with its likely effects on the economy as a whole. The study is based on the responses collected using a well-structured questionnaire.

Changes in the consumer's needs, desires, habits, standard of living and income levels have brought about a drastic change in the thinking of marketers and business firms. With the rise of consumer-oriented marketing, product is developed to satisfy the needs of the consumers. And as the needs of the consumer changes, business firms will have to reshape their products to suit the changing consumption patterns. As a result, business firms resort to product innovation. They either create a new product to satisfy the changed set of needs or they improvise their existing products to better satisfy their target market. Hence, an attempt has been made in this paper to understand the conceptual framework of product innovation, its meaning, types, dimensions and the key antecedents tofirm's innovativenessby reviewing the previous studies conducted in the related subject matter.