eChapters

Principle

Determination of the precise sequence of nucleotides in a sample of DNA is called “DNA Sequencing”. The most popular method for doing this is called the “Dideoxy method or Sanger Method” (named after Frederick Sanger, who was awarded the 1980 Nobel Prize in chemistry for inventing it). DNA is synthesized from four deoxynucleotide triphosphates. The top most figure is a normal nucleotide: deoxythymidine triphosphate (dTTP). During polymerization each new nucleotide is added to the 3' -OH group of the last nucleotide added.

The dideoxy method has been coined after the synthetic nucleotides that lack the –OH at the 3’carbon atom of the sugar and it plays an important role in this process atom (red arrow). In the figure below dideoxythymidine triphosphate — ddTTP, a dideoxynucleotide has been shown that can be added to the growing DNA strand. But the chain elongation ceases when the next nucleotide tries to get attached to the former as there is no 3' -OH for the next nucleotide to be attached to. Hence, the dideoxy method is also called the chain termination method. 

Identification of DNA as the Genetic Material

Experiments with pneumococcus suggested that DNA is the genetic material

Hershey and Chase provided evidence that the genetic material injected into the bacterial cytoplasm is T2 phage DNA

RNA functions as the genetic material in some viruses Work Before Watson and Crick

Nucleotides are the building blocks of nucleic acids

Nucleotides are linked together to form a strand

In 1869, Miescher first isolated DNA and named it “nuclein.”

In 1902, Archibald Garrod linked heredity to enzyme (protein) defects. In 1909 he published a book entitled “Inborn Errors of Metabolism.”

In 1928, Griffith identified a “transforming factor” that transmitted infectiousness in bacteria.

In 1944, Avery, MacLeod and McCarty showed that the transforming factor was DNA)

In 1950, Hershey and Chase, using their famous “blender” experiment, confirmed that DNA, not protein, is the genetic material.

The work of chemist Phoebus Levene showed that DNA includes deoxyribose, nitrogenous bases, and phosphates, in equal proportions.

Chargaff s data showed that the number of purines equals the number of pyrimidines.

Using Rosalind Franklin’s X-ray diffraction patterns, Watson and Crick deduced that DNA is a double helix

A laboratory is essential for research work in science and development. Most of the research institutes have dedicated laboratories for researchers and students to conduct experiments, perform research work, as well as to provide hands-on learning/training to students. There are certain rules and regulations laid down by the laboratory in-charge which need to be followed strictly for safety purposes. Some of the laboratories keep ‘safety manual’ for researchers to read before entering into the laboratory to conduct experiments. Whenever safety is compromised in a laboratory, there is increase in risk of unwanted incidents in the laboratory. Storing food/beverages in the same refrigerator where laboratory chemicals are kept may lead to disastrous effects on health. This chapter aims to provide instructions in brief on the things to be performed (Do’s) before and after entering onto a laboratory as well as the things never to be done (Don’ts) in a laboratory for the safety reasons.

Good Laboratory Practices (Do’s)

• P Wear apron/labcoat while working in the laboratory.
• P Use gloves/respiratory mask/fumehood while handling toxic/volatile/inflammable chemicals/reagents.
• P Use pippet or pippetor for safety in transfer of solutions.
• P Protect eyes, face and naked parts of the body from UV radiations. Use safety goggles or face mask/apron.
• P Keep your eyes away from Liquid Nitrogen and use cryogloves while handling it.
• P Arrange all the necessary materials/solutions before starting the experiment.
• P Periodically fumigate/ozonize culture and inoculation rooms.
• P Autoclave all contaminated materials before discarding.
• P Pre-treat gel or solution containing Ethidium Bromide (EtBr) before discarding into laboratory waste-disposal bin, and use gloves while handling them.
• P Burn all the transgenic plant materials in an incinerator after taking necessary observations.
• P Make entry in the designated log-book and clean the instrument after using it properly.
• P Switch-off the instrument after use or when not in use.
• P Wash hands with soap and remove the labcoat before leaving/going out of the laboratory7.
• P Ensure that switches for fans/lights, and water taps are off while leaving the laboratory.
• P Follow the biosafety guidelines suggested for handling and disposal of the hazardous chemical.

The full name of an organism technically has eight terms. For the dog it is: Eukarya, Animalia, Chordata, Mammalia, Carnivora, Canidae, Canis, and lupus. Notice that each name is capitalized except for species, and the genus and species names are italicized. Scientists generally refer to an organism only by its genus and species, which is its two-word scientific name, or binomial nomenclature. Therefore, the scientific name of the dog is Canis lupus. The name at each level is also a taxonomy Carnivora is the name of the taxon at the order level; Canidae is the taxon at the family level, and so forth. Organisms also have a common name that people typically use, in this case, dog. Note that the dog is additionally a subspecies: the “familiaris” in Canis lupus familiaris. Subspecies are members of the same species that are capable of mating and reproducing viable offspring, but they are separate subspecies due to geographic or behavioural isolation or other factors. Dogs, which are under the species Canis lupus familiaris, are known to have a total of 78 chromosomes (2n). The diploid genes can be classified into 38 pairs with two sex chromosomes.

Domestication of Livestock

Domestication (from Latin domesticus) is the process whereby a  population  of  animals  or plants is changed at the genetic level through a process of selection, in order to accentuate traits that benefit humans. It differs from taming in that a change in the phenotypical expression and genotype of the animal occurs, whereas taming is simply the process by which animals become accustomed to human presence. In the Convention on Biological Diversity, a domesticated species is defined as a “species in which the evolutionary process has been influenced by humans to meet their needs.” Therefore, a defining characteristic of domestication is artificial selection by humans. Humans have brought these populations under their control and care for a wide range of reasons: to produce food or valuable commodities (such as wool, cotton, or silk), for types of work (such as transportation, protection, and warfare), scientific research, or simply to enjoy as companions or ornaments. Plants domesticated primarily for aesthetic enjoyment in and around the home are usually called house plants or ornamentals, while those domesticated for large-scale food production are generally called crops. A distinction can be made between those domesticated plants that have been deliberately altered or selected for special desirable characteristics and those plants that are used for human benefit, but are essentially no different from the wild populations of the species. Animals domesticated for home companionship are usually called pets while those domesticated for food or work are called livestock or farm animals.

Donkeys can also be termed as ass (Equus africanus asinus) and is a domesticated member of the horse family, Equidae with chromosome number 62. The Wild ass of Africa, E. africanus is the wild ancestor of the donkey. For approximately 5000-7000 years, the donkey has been used as a working animal.

An adult male donkey is a jack or jackass, an adult female is a jenny or jennet, and an immature donkey of either sex is a foal. Jacks are often mated with female horses (mares) to produce mules, the less common hybrid of a male horse (stallion) and jenny is called hinny. NBAGR-ICAR has registered three breeds of donkey till date.

1. Kachchhi

• These donkeys are found in Kachchh district of Gujarat.

• Coat colour is mainly grey (dorsal surface grey and ventral surface white) followed by white, brown and black.

• Forehead is convex. Nasal bone is straight.

• Height at wither ranges from 77 to 115 cm.

• Docile in temperament.

• Only donkeys are used for agricultural purposes like inter cultivation for weed removing.

• Also utilized for transportation as donkey cart, as pack animal during pastoralist migration, etc.

The main purpose of this lecture note is to provide basic information on patent drafting skills and train applicants for drafting a patent in clear and concise manner.

We will see the components of Complete Specification through the following Case studey (1) of a solar water heater. The figures are provided below for reference and better understanding.

Abstract

Drug design is a complex computational process that involves studying the structural and physicochemical properties of biological moieties in order to identify new chemical or biochemical substances that can be used as drugs with specific pharmacological properties. The drug discovery pipeline is a crucial aspect of drug design that requires a detailed understanding of the various phases involved. In this chapter, we discuss the fundamental principles and phases of drug design. Additionally, we delve into the concept of docking, a critical step in the drug discovery process that involves predicting the orientation and affinity of a ligand binding to a protein target. The use of computational tools and techniques in drug design has revolutionized the field and is essential for the development of safe and effective drugs.

Tissue Collection

Rats are most commonly employed as test subjects to conduct the experiments related to drug metabolizing enzyme assays involving various enzymes i.e. microsomal and non microsomal enzymes. Rats are humanely sacrificed at the end of the exposure period of 28 days of any therapeutic agents or drug and the liver of all the animals has to excise and immediately wash with ice-cold normal saline, weigh and perfuse with ice-cold 1.15% KCl solution containing 0.05 mM EDTA. Livers are processed further for preparation of microsomes for determination of microsomal drug metabolizing enzymes.

Preparation of Microsomes

Livers tissues are homogenized in 3 volumes (1:3) of ice-cold (1-4o C) homogenization buffer containing 1.15% potassium chloride in 50 mM potassium phosphate buffer solution (pH 7.4). The homogenates are centrifuged at 15000 g for 30 min (Hitachi cs120GX II). The mitochondrial supernatant (8 ml) is transferred to ultracentrifuge tubes and spun at 105000 g for 1 hour in ultracentrifuge to sediment the microsomal pellet. Cytosolic fractions are collected from the middle separating the lipid layer on top in separate micro centrifuge tubes. The microsomal pellet is then washed by adding 8 ml of resuspension buffer (homogenization buffer containing 0.05 mM EDTA) and spun again 105000 g for 15 min. The microsomal pellet so obtained is resuspended in 4 ml of resuspension buffer and 1 ml aliquots are stored immediately at -80o C until assayed for drug metabolizing enzymes.

Introduction

While experimenting with chemicals, sometimes we need to dry the chemical/ solution (solid, liquid or gas) using a process like evaporation or distillation, mainly to remove water or different solvents. Drying is the process of evaporation or distillation to remove water/solvent from a solution, solid or even from gas. Generally, a drying agent (usually an anhydrous inorganic salt) is used for this process, which reacts with the water (present in the chemical/ solvent to be dried) to form a hydrate. For example, anhydrous MgSO4 is one of the drying agents. The drying agents are recognized by their capacity (the rate at which they absorb water) and their intensity (the amount of water left behind in the solvent at equilibrium). No chemical reaction or change in phase occurs during the drying process.

Type of Solvents

Apart from water (a universal solvent), there are three families of organic solvents used in laboratories.

1) Oxygenated solvents: Alcohols, Ketones, Glycol ethers, Glycol esters, etc.

2) Hydrocarbon solvents: Aliphatic and Aromatic hydrocarbons.

3) Halogenated solvents: Chlorinated hydrocarbons.

Computers take on an increasingly important role in today’s world. In this regard, their utility as tools for imparting high quality professional knowledge to veterinarians is much needed. e-learning covers a wide set of applications and processes, including computer-based learning, web-based learning, virtual classrooms, and digital collaboration. The delivery of content could be via electronic media, including the internet, intranets, extranets, satellite broadcast, audio/video tape, TV and CD-ROM. Information and Communication Technology (ICT) in the form of on-line teaching, face-to face video-cases and video-performances has recently been applied in the veterinary profession all over the world. ‘m-learning’ is the new buzz word almost indicating learning “anywhere” using mobile phones and Bluetooth or WiFi technologies. However, in the veterinary profession, connectivity is an issue as high quality network service is lacking in most remote areas. Some other terms frequently interchanged with e-learning are online learning, online education, distance education, distance learning, technology-based training, web-based training and even computer-based training (generally thought of as learning from a CD-ROM). e-learning is a broad term used to describe learning done at a computer.

Weaning to 210 days: Weight in kg of feed consumed for producing one kg of live weight for the period from weaning to 210 days of age.
10 Mortality percentages (as percentage)
Pre-weaning mortality: No. of piglets died during the period birth to weaning (28/56 days) the number of piglets born alive.
Post weaning mortality: Number of piglets died during the period from 28/56th to 365 day to the total number of piglets available at weaning.
Adult mortality: Number of adult pigs died to the number started at one year (in percentage).
List of registers to be maintained:
• Birth register
• Youngstock register
• Adult stock register
• Disposal register
• Mortality register
• Weighment register
• Farrowing and growth record
• Service register
• Boar performance register
• Sow performance register
• Veterinary register
• Feed efficiency register
• Account register

There are certain inherited traits (characters) in various species of livestock which are economically important. These traits have to be improved for increased production like milk, meat, egg, wool, etc.

Disruption of the cellular or anatomical continuity of the normal organ structure is known as wound. Healing involves migration, infiltration, proliferation and differentiation of several cell types like keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets which are culminate as an inflammatory response, new tissue formation and wound closure. Healing of the wound starts from the moment of the injury and can continue for varying periods of time depending on the extent of wound. The process of wound healing involves coagulation, inflammation, formation of granulation tissue, matrix formation, remodeling of connective tissue, collagenization and acquisition of wound strength.

Chitosan is a -1, 4-linked polymer of glucosamine (2-amino-2-deoxy—D-glucose) and lesser amounts of N-acetylglucosamine. It is a derivative of chitin (poly-Nacetylglucosamine) which is the second most abundant biopolymer after cellulose. Chitosan is first discovered in 1811 by Henri Braconnota, a French chemist and pharmacist. Later in the century, chitin is found in crustaceans (such as crabs, lobsters, shellfish and shrimp). Chitosan is a biodegradable polymer and it accelerates wound healing. It has been reported that chitosan permits regeneration of tissue elements in skin wounds and has positive application effects on wound healing.

Full thickness burn wound will not heal spontaneously which need early excision and adequate coverage to prevent fluid loss and risk of infection. Major portion of the skin is lost during burn injury which is the active layer for protecting the entry of micro organism from outside environment. So far, meshed autologous skin graft is regarded as the gold standard for treating extensive full thickness burns. However, their frequent use may be hampered by several impediments such as limited availability, donor site morbidity, associated with additional scarring. Severe burn patients invariably lack additional skin donor site. Moreover, creation of donor site in burned patient is equivalent to second degree burn and which further increase the total body surface area affected (TBSA). In past decades, numerous biomaterials have been investigated for the use as a dermal skin substitute for burn patients but none give promising results. The innovative tissue engineered bioactive materials offer potential tissue regeneration and prevent the tissue deterioration.

The extracellular matrix (ECM) derived naturally from mammalian tissue have been successfully used as scaffold for tissue reconstruction and wound management in clinical applications. ECM can be derived from various tissue or organs of different species but most commonly, porcine small intestinal submucosa and urinary bladder have been successfully used as scaffold for reconstruction. ECM is obtained by process of decellularization that involves the use of various ionic and non ionic detergents, acids, enzymatic solutions and mechanical/physical methods.

Introduction

Proper electrical wiring and earthing are crucial for laboratory safety and functionality. A laboratory houses several installations/equipment that demand stable/continuous power supply. Inappropriate or faulty wiring and earthing may lead to significant risks, including electrical shocks, damage to equipment, and even incidence of fire. Therefore, understanding the requirements of electrical wiring and earthing is essential for designing, construction, and maintenance of laboratory. This chapter aims to provide a basic knowledge of electrical wiring and earthing necessary for a laboratory. Importance of earthing in mitigating electrical hazards and ensuring integrity of electrical installations are also described.

Basics of Electrical Wiring

Basic understanding of electrical wiring is necessary for laboratory safety and efficient electrical functioning. The potential difference between two electrical points (negative and positive) is known as Voltage, which drives the flow of electric current. This is measured as volt (V). The rate of flow of electricity in a circuit is known as Current (I), which is measured as ampere (A). Current can be either direct (DC) or alternating (AC). Opposition/resistance experienced in the flow of current in circuit is known as Resistance (R) and it is measured in ohms (Ω). Higher the resistance means lesser the current flows at a given voltage. The rate at which electrical energy is transferred/utilized in an electric circuit is known as Power (P), which is measured in watt (W). The Power can be calculated as the product of voltage and current (P = V × I).

Principle

To retrieve and purify any specific DNA fragment from an agarose gel slice; the expected yield is from 50-75% of the amount in the gel slice.

Time required

Restriction digest - minimum of 2 hours

Electrophoresis - approx. 4 hours

Elution - 2-5 hours depending on fragment size

DNA Purification -2 hours

Principle

The principle of Electrophoresis involves the motion of a particle in a fluid medium, where the gravitational attraction (on a flowing liquid) is the sedimenting force, and the frictional force is the opposing force and is proportional to the velocity of the particle. These forces balance each other acting in opposite directions and hence balance each other. This will lead to the uniform motion of the particle in the mobile liquid medium (i.e. the particle moves at constant velocity). In case the external field is an electric field instead of a gravitational field, a macromolecule will respond to the external field in two ways. In case the molecule is charged, it will migrate in an electric field to the electrode of opposite charge. Under this principle the technique of electrophoresis functions.

All electrophoresis operations are governed by the principle:

Introduction
The urinary system comprises of two kidneys, two ureters, a urinary bladder, and a urethra) The latter is shared by the genital organs.
• The urinary system develops from the intermediate mesoderm
• Three different kidney systems are formed in vertebrates--pronephros, mesonephros, and metanephros.
Pronephros: - Forms a duct that empties into the cloaca- Pronephric tubules connect each body segment with the coelom (body cavity) Pronephric tubules never become functional in mammals
Mesonephros: - Pronephric duct becomes mesonephric duct Distal to the pronephric tubules appear mesonephric tubules that surround tufts of blood vessels (glomeruli), but no loop of Henle is formed Blood is transported to mesonephric tubules, filtrate is transported to the mesonephric duct and finally to the urogenital portion of the cloaca
Metanephros: - Metanephric bud (ureteric bud) Metanephric blastema
• Bud gives rise to the ureter, the pelvis of the kidney, and collecting tubules.
• Blastema gives rise to nephrons.
The internal structure of the kidney varies among species. It is determined by the number and size of calyces which branch from the renal pelvis. Each calyx forms the base of a renal lobe (renal pyramid). All mammalian kidneys begin as multipyramidal (multilobular) structures, but lobules fuse to varying degrees, depending upon the species. (Note, this multilobulation is maintained in the histological structures)

Technology Scouting and Innovations in Technology Incubation
Technology scouting and innovations in technology incubation have a symbiotic relationship, fuelling each other for the benefit of budding startups and the broader technology ecosystem. Let’s dive deeper into their individual roles and how they interact:
Technology Scouting
Technology Scouting is the process of discovering, analyzing and evaluating new or existing technologies that will help them with their innovation process. In simple terms, it is a process for companies to find the necessary technology outside the company.
Hunting for promising technologies: Scouts actively search for emerging technologies, patents, research advancements, and market trends across various sectors.
Evaluating potential and fit: They assess the technical feasibility, market potential, and compatibility with the needs of incubators and their supported startups.
Connecting innovators and ventures: Scouts bridge the gap between technology creators (researchers, inventors, startups) and potential users (incubators, established companies).
Fuelling innovation pipelines: Technology scouting helps incubators access a wider range of technologies and ideas, enriching their portfolio and diversifying the innovations supported.