eChapters

Amino acids serve as the building blocks of proteins and as precursors of many other important biomolecules such as hormones, purines, pyrimidines, porphyrins and some vitamins. They also serve as sources of energy when present in excess amounts and when they are used as fuel, amino acids undergo deamination reactions. The remaining carbon skeleton will either convert itself into glucose or undergoes oxidation to CO₂ via TCA cycle.

Central dogma: The Central dogma of molecular biology, which has been formulated as a simple linear progression of information from DNA to RNA to Protein, is summarized in the illustration. The replication process on the left consists of   passing  information from a parent DNA molecule to daughter molecules. The middle  transcription process copies this information to a mRNA molecule.  Finally, this information is used by the chemical machinery of the ribosome to make polypeptides.

Introduction

Proteins are the most abundant organic compounds constituting major part of the body. Chemically, proteins are nitrogen containing macromolecules and polymers of L α-amino acids, which serve a wide range of structural and biological functions in animal body. For example, connective tissue made up of collagen, metabolicenzymes, neuro transmitters,signal in gmolecules, structural components of cell membranes and cell organelles, etc. all are chemically proteins.

Before goinginto the details of protein metabolism, first, we need to understand that there are majorly two sources of proteins in our body: dietary protein and body protein. Dietary protein is taken in diet, while body protein indicates the protein and amino acids synthesized in animal body to fulfill the requirements for growth and development. The digestion of proteins will not be discussed here as it has been described in detail in chapter 12. Dietary protein is digested in presence of several enzymes such as trypsin, pepsin, chymotrypsin, aminopeptidases and carboxypeptidases and is converted into mono-, di- and oligopeptides in stomach and small intestine; and finally converted into amino acids. These amino acids are transported to the systemthrough intestinal mucosa via active transport, from where they enter into the cells for further metabolism. Therefore, ‘metabolismof proteins’ is more appropriatelylearnt as ‘metabolism of amino acids’.

Summary

Researchers face a series of similar-yet unique problems to isolate proteins. To solve these they must have dip knowledge to select an appropriate technique. The mechanism, to study the way in which a living system works, is necessary to dismantle the machine and to isolate the component parts so that they may be studied, separately and in their interaction with other parts. The knowledge that is gained in this way may be put to practical use, for example, in the design of medicines, diagnostics, pesticides, or industrial processes. In every case a pure protein is desirable as impurities may either be misleading, dangerous or unproductive, respectively. Protein isolation is, therefore, a very common, almost central, procedure in biotechnology. The purpose of this chapter is thus to fill the beginners with techniques relevant to protein isolation, hopefully to improve their knowledge.

Introduction

All the amino acids have in common an amino group and a carboxyl group attached to the -carbon atom; however, they differ in having distinctive side chains, usually called R groups, as is shown in the following  generalised structural formula

Abstract

The field of bioinformatics emerged as a tool to facilitate biological discoveries more than 10 years ago. Structure prediction is fundamentally different from the inverse problem of protein design. Protein structure prediction is one of the most important goals pursued by bioinformatics and theoretical chemistry; it is highly important in medicine and biotechnology. Structure prediction is fundamentally different from the inverse problem of protein design. While the genome of many organism have been sequenced over the last few year, transforming such raw sequence data into knowledge remains a hard task. A great number of prediction program for genes have been developed hat try to address this problem, which consists of locating the genes along a genome.

1. Introduction

Protein–protein interactions serve as the hub of the functioning of a living cell, coordinate biological processes as well as form core for interactomics system of living organisms. Various biological processes as signal transduction, cell-cell communication, ingestion or expulsion of any macromolecule, host parasite interaction, diseases as cancer and many other metabolic networks employ protein–protein interactions. During such a process two or more proteins interact transiently with each other or form complex for a long period of time. Thus attempts have been made for years to study comprehensive mechanism of such protein–protein interactions by various branches of sciences such as biochemistry, quantum chemistry, molecular dynamics, chemical biology and signal transduction. The availability of three-dimensional (3D) structure of proteins is a prerequisite for understanding functions of protein complexes at a molecular level as well as amino acid interactions within a complex.

Abstract

The field of bioinformatics emerged as a tool to facilitate biological discoveries more than 10 years ago. The ability to capture, manage, process, analyze and interpret data became more important than ever. Bioinformatics in agricultural science has many complex aspects and problems that must be resolved due to the involvement of a wide variety of fields and subjects. Consequently, the establishment of interdisciplinary research areas is required, which should include collaboration among experimental and informatics researchers in different fields within industry, academia, and government. Thus, the Agricultural Bioinformatics Special Interest Group was established to promote interaction among researchers and provide an opportunity to report on bioinformatics studies in agricultural science: design and analysis of functional foods, scientific testing of food safety, breeding of agricultural organisms, development of environmental friendly agricultural chemicals, bioremediation and production of useful chemical materials using microorganisms, measuring and monitoring biodiversity, statistical genetics in resource ecology, and so on. The activities of the Agricultural Bioinformatics Special Interest Group include (1) Studies of applied bioinformatics in the agricultural science (2) Development of new research areas in agricultural science using bioinformatics (3) Practical application of bioinformatics technologies in the field of agricultural science (4) Development of new bioinformatics technologies characteristic of agricultural science.

Abstract

Proteins are complex molecules composed of one or more polypeptide chains made up of amino acid residues. These molecules perform a variety of essential functions in living organisms, including DNA replication, molecule transport, catalyzing metabolic processes, and providing structural support to cells. In this chapter, we explore the four levels of protein structure: primary, secondary, tertiary, and quaternary. Primary structure refers to the specific sequence of amino acids in a polypeptide chain, while secondary structure describes the folding of the chain into alpha-helices, beta-sheets, or other patterns. Tertiary structure is the three-dimensional shape of a single polypeptide chain, and quaternary structure refers to the interactions between multiple polypeptide chains to form a larger protein complex. Additionally, this chapter covers various methods used to predict protein structure, including homology modeling, ab initio prediction, and threading algorithms.

Abstract

Protein-protein interactions play a major role in most cellular processes. Many essential cellular processes such as signal transduction, transport, cellular motion and most regulatory mechanisms are mediated by protein-protein interactions. The study of protein-protein interactions is of great importance in determining the biological function and impact of proteins. The experimental techniques remain expensive, time-consuming and labor-intense. Computational methods play an important role at all the stages of determining protein-protein interactions. In this chapter, we describe the different types of protein-protein interactions, computational methods, currently available databases and tools, protein-protein docking and as well as protein-protein interactions as drug targets.

Proteinase inhibitors (PIs) are small proteins that are quite common in nature. They are natural, defense-related proteins often present in seeds and induced in certain plant tissues by herbivory or wounding (Koiwa et al., 1997). PIs are present in multiple forms in numerous tissues of animals and plants as well as in microorganisms. In plants they can be counted among the defensive mechanisms displayed against phytophagous insects and microorganisms. The defensive capacities of plant PIs rely on inhibition of proteases present in insect guts or secreted by microorganisms, causing a reduction in the availability of amino acids necessary for their growth and development. PIs are of common occurrence in the plant kingdom. Plant PIs (PPIs) are generally small proteins that have mainly been described as occurring in storage tissues, such as tubers and seeds, but they have also been found in the aerial parts of plants (De Leo et al., 2002). One of the important defense strategies that are found in plants to combat predators involve PIs which are in particular effective against phytophagous insects and microorganisms. The defensive capabilities of PPIs rely on inhibition of proteases present in insect guts or secreted by microorganisms, causing a reduction in the availability of amino acids necessary for their growth and development (Lawrence and Koundal, 2002).

Proteins are large biological molecules, or macromolecules, consisting of one or more chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including catalysing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its activity. A polypeptide is a single linear polymer chain derived from the condensation of amino acids. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids; however, in certain organisms the genetic code can include selenocysteine and, in certain archaea-pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by post translational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes.

Like other biological macromolecules such as pollysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signalling, immune responses, cell adhesion, and the cell cycle. Proteins are also necessary in animals’ diets, since animals cannot synthesize all the amino acids they need and must obtain essential amino acids from food. Through the process of digestion, animals break down ingested protein into free amino acids that are then used in metabolism.

INTRODUCTION

Plants are rich source of nutrients for most of the organisms including microbes' arthropods and vertebrates. There are various biotic viz. insects, weeds and pathogens and abiotic factors viz. temperature, humidity, salinity etc. that causes harm to plants. For yield loss due to pest EIL (Economic injury levels) calculation is important (Stern et al, 1959). EIL calculation will define economic damage (yield losses equal to management costs) in calculable terms. Since plants are sessile they cannot move to avoid the menace of these factors, but they have developed a unique way of structural, chemical, and protein-based defences which are specifically designed to detect and inhibit invading organisms before they are able to cause extensive damage. In response to the incitations by the pathogens some genes are activated which in turn codes for defence proteins (Grennan, 2006). Understanding how plants defend themselves from pathogens and herbivores is essential in order to protect our livelihood and nutritional security. In addition to proteins, plant anatomy and some of the ecological relationship also plays an important role in plant defense mechanism. Induction of plant resistance against fungi and bacteria was reported in 1900.

The word protein was first coined in 1838 to emphasize the importance of this class of molecules. The word is derived from the Greek word proteios which means “of the first- rank”. Proteins are polymers/macro molecules made up of several amino acids. When there are more than 50 amino acids in a chain, the polypeptide is normally called a protein. Each protein in the cells of living beings has a unique sequence of amino acids that determines its biological function. The proteins are folded into specific defined structures, which are maintained by a large number of relatively weak bonds. The  three  dimensional structure is well defined and suited to it’s specific function.

Very small changes in the structure can modify the function. Hence, it is important to know the structure of proteins to understand their behavior and function. The biological activity of proteins depends on maintenance of folded conformation. Proteins fold into well defined three dimensional shapes and they are able to recognize their corresponding substrates or antigen molecules and bind them tightly. The protein structure has been classified into four different levels based on the folded conformation of the protein.

1.    The basic building block(s) of proteins is/are

(a) Amino acids

(b) Phenolics

(c) Carboxylic acids

(d) Fatty acids

2.    Alpha helix and ?- pleated sheet are characteristic of protein structure?

(a) Primary

(b) Secondary

(c) Tertiary

(d) Quaternary

1. INTRODUCTION

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. Proteins are the important constituents of foods for a number of reasons. They are a major source of energy, as well as containing essential amino-acids, like lysine, tryptophan, methionine, leucine, isoleucine and valine which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability.

Proteins are complex organic molecules made up of carbon, hydrogen, oxygen, and nitrogen. Most proteins also contain sulphur and phosphorus and also play an important role in food preparation. The final quality of many food products are influenced by how the proteins are treated while food is being prepared.

Proteins are composed of amino acids and are large molecules that play a crucial role in various biological processes. They are considered the fundamental building blocks of life due to their abundance in the body, constituting approximately 60% of the dry weight of cells. These complex biomolecules are intricately folded and twisted strands of amino acids, actively participating in vital life processes, including metabolism, locomotion, immune response intercellular signaling, and molecular identification. As macronutrients proteins are present in all living organisms and directly contribute to numerous metabolic pathways. It is important to note that proteins are unique to each organism, exhibit species specificity, and vary within different organs of the same organism. With 22 different amino acids, the properties of a protein molecule are determined by the specific arrangement of these amino acids. While plants can synthesize all the necessary amino acids for protein production, animals rely on external sources for certain amino acids. Proteins consist of amino acids connected through peptide bonds, formed by the linkage between the amino (NH2 ) group of one amino acid and the carboxyl (COOH) group of another amino acid.

Introduction

Aquatic ecosystems are critical components of the global environment and provide a variety of resources for human population, including food, water for drinking and irrigation, recreational opportunities, and habitat for economically important fisheries. Fish is the main source of animal protein for 3 billion people worldwide, providing a valuable protein complement to the food platter worldwide. Fish is also an important source of essential vitamins and fatty acids. Furthermore, it provides an important source of cash income for many poor households and holds a great potential as a source of foreign exchange. However, aquatic ecosystems are increasingly being threatened directly and indirectly by climate change. Global warming with consequent climate variability and extreme weather conditions has a great impact on the aquatic ecosystems resulting in increased temperature variation, ocean acidification and freshwater salinization etc. As per report of IPCC 2018, global warming of 1.5 ºC above pre-industrial levels has been declared (IPCC, 2018) and expected to rise further in future. Thus, variations in temperature, salinity, acidity and dissolved oxygen along with the effects of other anthropogenic activities may exert strong in?uences on aquatic ecosystems and the aquatic animls will be facing these climate-related stressors in near future. Such environmental stressors is projected to alter fundamental ecological processes, geographic distribution of aquatic species, reproductive and immunological performance of species etc.

The genera Proteus and Providencia belong to the tribe Proteae of the family Enterobacteriaeceae. The members of both these genera are gram negative, motile bacilli, aerobes and facultative anaerobes and can grow on basic media. A characteristic feature which distinguishes tribe Proteae from other members of Enterobactereaceae is the presence of the enzyme phenylalanine-deaminase which converts phenylalanine to phenylpyruvic acid (PPA reaction).They also produces a powerful urease enzyme which rapidly hydrolyses urea to ammonia.
Genus Proteus
They are gram-negative bacilli, 1-3 μm long and 0.6 μm wide. They are noncapsulated and are actively motile by peritrichous flagella. The name ‘Proteus’ refers to their pleomorphism, after the Greek God Proteus who could assume any shape. Four species: Proteus mirabilis, P.vulgaris, P.penneri and P.myxofaciens are recognized. Proteus mirabilis, P.vulgaris are widely recognised as human pathogens.
Culture Characteristics
These can grow on ordinary media like nutrient agar with a characteristic fishy or seminal odour. On MacConkey and Teepol lactose agar, lactose nonfermenting pale colonies, around 2-3 mm in size are formed. On non-inhibitory solid media such as blood and nutrient agar Proteus mirabilis and P vulgaris show characteristic swarming growth in the form of a uniform film, which spreads over the whole surface of the plate. In young swarming cultures, many of the bacteria are long, curved and filamentous, sometimes reaching upto 80 μm in length. When two different strains of swarming proteus mirabilis encounter one another on an agar plate, swarming ceases and a visibile line of demarcation forms. This is known as the Dienes phenomenon. In liquid medium (peptone water, nutrient broth), Proteus produces uniform
turbidity with a slight powdery deposit and an ammoniacal odour.

1.    Collect the bone marrow from iliac crest of rabbit, dog, goat and sheep; sternum of pigs by 16-18 G bone marrow biopsy needle under sedation/anesthesia and maintain strict asepsis protocol
2.    To aspirate bone marrow, negative pressure should create in the syringe by pulling back the plunger. Syringe should be first flushed with heparin before bone marrow collection
3.    Collect approximately 2.5-5 ml of bone marrow from each animal
4.    Mix the bone marrow sample with equal volume of low DMEM having 10% FBS and 1 ml of PS and then layer on 5 ml of histopaque/Ficol-Paque Plus in a falcon tube
5.    Centrifuge at 3000 rpm for 30 min
6.    Collect stem cells from the interface with the help of a micropipette
7.    Cells wash thrice with DPBS 
8.    Centrifuged at 2000 rpm for 5 min

Collect the femur and tibia and clean them very well from blood, tendons, muscle, skin and other tissues (using scalpel)

Cut off epi-and metaphysis from the long bones with electric/manual bone saw
Transfer the open bones to a falcon tubes containing cell culture medium/DPBS without FBS but containing 1% PS (Penicillin-streptomycin)
Transfer the bones to a Petri dish containing 5 ml medium without serum (FBS) and without antibiotic (40-50 mm Petri dish)
Remove the pteri dish to the laminar cabinet
Collect the bone marrow by flushing out to a 50 ml falcon tube (use syringes and needles to flush out the bone marrow; flush out the bone marrow with complete (10% FBS, 1% PS, 90% DMEM-LG) culture medium
Centrifugation: 300rpm, 8 min (in the meanwhile prepare 5 ml of Ficoll into a 15ml falcon tube)
Aspire the medium

1.    Collect fat tissues from euthanized animals into a Falcon tube containing DPBS + 1% PS ( for transferring lab to lab)
2.    Wash 3 times with DPBS + 1% PS
3.    Inside a laminar cabinet, transfer the fat to a Petridis (use an sterile spatula to collect the fat)
4.    Cut the fat into small (mm) pieces using scalpel and forceps or scissors
5.    Transfer the fat into a Falcon tube (eg into a 50 ml Falcon tube you can add upto 10 ml fat tissues)
6.    Wash the fat by adding approx 40 ml DPBS; Centrifugation: 430g, 10 min (without break)
7.    Transfer the fat into a fresh 50 ml Falcon tube (with the help of sterile spatula); Repeat steps 5 to 7 for 2-3 times

Characterization of Oct-4/Sox-2 Transcription Factors by Immunostaining for Characterization of Stem Cells
Protocol
1) The cultured cells at a density of 70,000/well in a 6 well plate or 35,000/ well in a 12 well plate are used for immunostaining.
2) The cells are washed with Dulbecco’s phosphate buffered saline (DPBS) for two times and the cells are fixed using 4% paraformaldehyde for 20 minutes at room temperature.
3) After fixation, the spent Paraformaldehyde is removed and the fixed cells are washed with 1000μl of 1x DPBS for 2 times by clockwise and anti-clockwise rotation of the plate slowly.
4) About 400μl of 3% blocking buffer was added to the wells and plate was covered with aluminum foil to prevent the exposure of light and stored at room temperature for 90 minutes.
5) After blocking, the cells are again washed once with DPBS. About 500 μl of primary antibody Oct-4 in the dilution of 1:40 is added to the well and stored at 4°C over night.
6) After overnight staining with primary antibody, the antibody solution is removed from the wells and washed with DPBS for 2 times.
7) Take 500 μl of secondary antibody anti-rabbit-FITC diluted with antibody buffer solution is added in the ratio of 1:200 and cover with aluminium foil and incubated for 90 minutes.
8) Secondary antibody is removed and washed with DPBS.

1.    Remove the growth media
2.    Wash 2 times with warm PBS (w/o Ca and Mg)
3.    Add 5ml of trypsin /EDTA 0.35 % (Invitrogen), place at 370C in a CO2 incubator for 4-5 min
4.    Wait until cells are detached/lifted and make sure by examining on microscope
5.    Add equal volume (5ml-1:1) of growth medium as appropriate and mix with 5 strokes
6.    Transfer into 15 ml centrifuge tube
7.    Centrifuge at room temperature for 4 min in 980 rpm
8.    Remove supernatant complete but carefully without touching the cell aliquot
9.    Re-suspend in 10ml DPBS (with Ca and Mg)-5 strokes
10.    Immediately after mixing transfer 100µl of cell suspension into Casy vial, containing 10ml Casyton

1. Fix the organ or the animal in 5-10 % formalin preferably neutral buffered formalin. If the organ is an isolated thoracic limb, for example, then cannulate the axillary artery and perfuse the formalin. I find sometimes it is advantageous to pump some buffered saline solution to flush out any blood before pumping in the formalin. Organs, such as the heart, could be nicely flushed with water through major blood vessels, and immersed in formalin to fix. The same can be done with liver, kidney, etc. It takes a week for fixing a large structure (e.g., 10 kg) by immersing in formalin.
2. Dissect the specimen as needed. Remove all fat and clean as much as possible so the specimen looks nice.
3. If the specimen is dark brown, it can be lightened by leaving 1-2 days in 5% hydrogen peroxide. Check the specimen 1-2 times during the day to make sure desired light tone is achieved. Leaving it too long in the hydrogen peroxide may result in tissue degradation.
4. Drain the specimen well, and dehydrate in acetone. The dehydration should be thorough. To achieve this, use 100% acetone, at least 2-3 changes. The acetone can be recycled by repeated distillation. Acetone tends to evaporate, so use a container that has a tight lid. Acetone is also flammable, so use required precautions when handling acetone. If concentration of acetone remains up to 99% even after leaving organ for 4-5 days it is sure that dehydration is complete. If you are using any thin sheets of organ or Brain for dehydration keep slice between glass plates and then dehydrate.
5. Immerse the specimen in glycerin. Buy industrial glycerin, which may be cheaper. It may take 2-3 weeks for the glycerin to permeate into the tissues.

6.1 Designing primers

Designing correct primers are very important to get the expected result in PCR experiment. There are two primers (Forward & Reverse) required for the amplification of the desired DNA fragment. One primer should anneal to the sense strand (oriented to the 5'→3' direction) and another primer should complement the antisense strand (oriented in the 5'→3' direction).

There are various characteristics to be considered to design a good primer including Tm, length, GC content, secondary structure and specificity.

There are commercially available softwares besides free online tools at NCBI (primer blast) for designing PCR primers. In addition, primer sequences published in peer reviewed literature can also be used.

For RT-PCR, the extracted total RNA is first reverse transcribed to cDNA using reverse transcriptase enzyme. Following are the protocols for cDNA sysnthesis and RT-PCR.

Note: cDNA synthesis and RT-PCR can be performed according to the manufacturer protocol from where the specific reagents for the above methodologies are purchased.

8.1 Purpose

To lay down a uniform protocol for the synthesis of c-DNA from total RNA

8.2 Scope

The scope of this Standard Operating Procedure is to lay down a standard procedure for the synthesis of c-DNA from total RNA

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows:

1. Preparation of primary cells culture as described in earlier methods.

2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method.

3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent.

4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.

5.1 Purpose

To employ a standard protocol to perform agarose gel electrophoresis.

5.2 Principle

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. Migration rate of DNA molecule depends on various factors like 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under UV light.

11.1 Principle

In situ hybridization techniques involve detection of specific nucleic acid sequences to in morphologically preserved chromosomes, cells or tissue sections. Along with immunocytochemistry, in situ hybridization can correlate microscopic topological information to gene activity at the level of DNA, mRNA or protein. The FISH protocol involves 1) denaturation and hybridisation performed on Day 1 and 2) washing and detection performed on Day 2. DNA of the chromosomes and the probes are denatured and allowed to hybridize overnight on the first day. On day 2, slides are washed to remove unbound DNA sequences followed by detection, counterstaining and mounting of the slides.

10.1 Purpose

To lay down a standard protocol to perform qPCR for quantitative analysis of target genomic sequence.

10.2 Principle

qPCR or quantitative polymerase chain reaction or real-time polymerase chain reaction, is a molecular biology technique to amplify and at a time quantify a targeted DNA molecule and based on the polymerase chain reaction (PCR). It is called real time as the amplified DNA is detected as the reaction progresses and holds an advantage over standard PCR where the reaction is detected at its end. There are two common methods in quantitative PCR to detect the products i) non-specific fluorescent dyes which intercalates with any double stranded DNA ii) sequence-specific DNA probes labeled with a fluorescent reporter consisting oligonucleotides. This reporters helps in detection of the complementary hybridization of the probe and quantifies amount of DNA in cells or tissues.

9.1 Purpose

To lay down a standard protocol to perform RT-PCR for quantitative analysis of target RNA.

9.2 Principle

RT-PCR technology is used to amplify a specific RNA sequence (target sequence) to measure its expression inside the cell. RT-PCR follows the same principle as of normal PCR, but instead of DNA here cDNA is used as template for amplification. Extracted RNA is reverse transcribed to cDNA with the help of reverse transcription enzyme. The cDNA is then subjected for PCR amplification with the help of DNA polymerase, target specific primers, nucleotides and reaction buffer. In molecular diagnosis, RT-PCR is used to detect the expression of oncogenes at the time of diagnosis. It is also used to detect minimal residual disease during treatment

Proto – first; Zoon – animal

Protozoa are single celled eukaryotic organisms, which lack cell wall and ability to move at some stage of their life cycle.

They are referred as the first animals.

There are about 65000 described species grouped into 7 phyla. Among them, 50 per cent are not available as living forms (as fossils) and out of remaining 50%, 22,000 species are free-living and 10,000 species are parasitic forms.

The protozoa are distinguished from other organisms by following characters

Introduction

The soil protozoa are unicellular and the simplest forms of animal life. Most of the protozoa are devoid of chlorophyll but transitional genera resemble the algae and possess chloroplasts containing chlorophyll pigments. The active life stage is commonly known as trophozoite phase where they feed and multiply but under adverse climatic conditions, they form resting stage or cyst, which develop a thick coating on the cell surface. Mostly, the protozoa multiply by longitudinal or transverse fission but some species reproduce sexually by fusion of two cells followed by exchange of genetic material between their nuclei and cell division.

Protozoa are single celled eukaryotes which are either parasitic or free living belonged to family apicomplexa. These protozoan affects the different birds like chickens, turkey, ducks, pigeons, game birds and also various pet birds. The infection caused by protozoan diseases may affect one system or more than one system of the body depending on the causative agents involved. The common protozoan diseases like leukocytozonosis, chochlosomiasis, cryptosporidiosis, coccidiosis, histoplasmosis causes infection in the ducks. The protozoan diseases cause severe economic loss to the poultry industry due to death of birds, expenditure on the treatment and prevention of disease and loss of egg and meat production.

Introduction

The protozoa (meaning proto- first and zoa mean animals) are considered to primitive and are unicellular organism. The protozoa are Eukaryotic organism having nucleus in which the genetic information is stored in chromosomes of nucleus. In poultry, the protozoan disease categorized into two main group

1. Intestinal protozoan parasites include Eimeria spp. (Coccidiosis), Cryptosporidia (Cryptosporidiosis) Histomoniasis (Histomonas meleagridis), Trichomoniasis (Trichomonas spp.) and Spironucleus (Hexamitiasis).

Coccidiosis is important disease of poultry caused by protozoa of the genus Eimeria. The disease occurs by ingestion of sporulated oocyst ehcih upon action of trypsin rupture in gizzard and proventriculus relealeses sporozoites. The sprozoites penetrate mucosa of intestine and two generation of asexual reproduction occurs (Schizogony) and releases merozoites which mature and produces gamonts. Sexyual reproduction occurs (Gametogony) and produses zygot which mature to form oocysts. The oocysts are passed in faeces. The high moisture (exceeding 30%), immunosuppressive diseses and environmental and managemental stress lead to occurrence of disease. The birds affected with coccidiosis show depression, ruffled feathers, blood tinged diarrhoea and pale comb and wattle. Specific part of intestine is affected by each species of Eimeria. Diagnosis of coccidiosis is done by finding oocyst in faeces under microscope.

1 Professor (Retired) Department of Agricultural Extension and Rural Sociology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu

2 Assistant Professor, Department of Agricultural Extension and Rural Sociology, Agricultural College & Research Institute, Madurai-625 104

Introduction

The word ‘Extension’ was first coined in U.S.A. in the mid-part of the nineteenth century, to connote extension knowledge of improved practices in agriculture and home economics from Land Grant Colleges to the farmers and their families in their fields and homes. The literary meaning of Extension is ‘Ex’-out; Tensio’-to stretch i.e. Extension means stretching out. Extension, in the context of rural community development, means educating rural people and it implies production of changes in human behaviour -in terms of attitude, knowledge and skill. Extension is often misconceived as disbursement of subsidies and providing certain material incentives; it is certainly not.

There are several conventional extension techniques approaches and tools for transfer of any farm -related technologies, be it any enterprise, crop or their mix. Extension teaching aids are broadly classified as:

The condition is characterized by proventriculitis and dilatation of proventriculus.

Etiology

The cause of transmissible viral proventriculitis (TVP) is a birnavirus referred as Chicken proventricular necrosis virus (CPNV). TVP is a disease of 3-8 week-old broiler.

Agricultural research in India has contributed new varieties of crops and production technologies for increased production at the national level. These modern technologies benefited only a particular section but farm youth in rainfed areas are facing many problems. One of the problems identified in the diffusion of technologies to farm youth in sustainable agriculture was the lack of appropriate technologies suitable to the farm and social situations. When farmers in rain fed areas are exposed to innovations, they react to the practices/innovations from different viewpoints. A practice appropriate to farm youth in rain fed areas must be less in cost, high in profits, less risky,  highly compatible with socio-cultural values and beliefs, less in complexity, highly trialable on a small scale and the results of the same should be easily observed and communicated. These dimensions are perceived by farmers differently and the degree to which they perceive them favourably are likely to adopt the same in rain fed areas. Many a time, several past investigations have only indicated whether the recommended package of technologies has been adopted or not by farm youth. This gives percentage of farm youth adopting the practices but not the percentage of recommended technologies adopted by them and the problems encountered by them in adopting the recommended technologies in rain fed areas.

Definition

Wound with infection with 6 hours of occurrence can be brought to non infected stage by flushing.

Procedure

The infected wound at fracture site is cleaned / flushed thoroughly using sufficient quantity with antiseptic solution.

The site is applied with topical antiseptic / antibiotic and the size of the wound is reduced by placing interrupted stitches for skin closure.

For drainage of inflammatory fluid, the counter opening is made as the distal part of the swelling of the dependent part of flushing catheter is placed inside to carry out regular flushing of the wound without removing the immobilizer.

The fracture site is immobilized as per the conventional procedure using POP cast leaving two window over the opening of counter opening site.

Traditionally, Labour Laws have a protective function consisting of established standards both to protect workers in their workplace and to provide them a basic minimum level of living conditions. Alongwith this function, there also existed in most systems, a legal framework for the regulation of industrial relations between employers and workers. These functions had their impact on problems of development. The emphasis was more on labour legislation for its own sake, development being a separate proposition. This mutual lack of concern was perhaps due to historical factors. The most important aspect is the fact that in developed countries of the Western world, labour legislation followed the emergence of industrialization and in response to a demand for economic and social betterment of the workers, whereas in developing countries the emergence of labour laws preceded industrial growth.

Proximate analysis: Weende’s system is followed. Weende was a small village in Germany, near the University of Goettingen at that time (1860), when this system was developed by Hanneburg and Stohmann. Although it is neither true nor approximate, but somewhere in the middle, that is, proximate analysis of the various parameters for assessment purpose; it gives information about the quality of feeding stuff and still followed.

General Views about Weende’s System of Analysis

A team of scientists under the leardship of Henneberg and Stohmann of (University of Göttingen, Germany) developed proximate principles analysis system in the sixties of last century at Weende’s Experimental Station (Germany), located at the University of Göttingen. Main components of different fractions in the proximate analysis of foods are moisture, ash, crude protein, ether extract, crude fiber, and nitrogen-free-extract. These workers emphasized that carbohydrates could be grouped into: (1) the starches and the sugars, and (2) coarse fibrous fractions. Later on an insoluble residue after boiling the fat free food sample first with dilute acid and then with dilute alkali was obtained based on the acid stomach digestion and the subsequent alkaline intestinal digestion of consumed food. At the end of digestion insoluble organic residue was denoted as crude fiber.

Two German scientists Henneberg and Stohmann working at the Weende Experimental Station, Germany devised proximate principles analysis system in 1860’s for routine description of animal feedstuffs. Therefore this system is also known as Weende system of analysis. Proximate analysis is a system of expressing the nutrient composition of a feed. The principle of analysis is to separate the nutrient present in the feedstuffs into various fractions, as proximate principles, which are six i.e. moisture, ash, crude protein, ether extract, crude fibre and nitrogen free extract.

Water (Moisture)

The moisture is determined as the loss in weight which results from drying a known weight of feed to constant weight at 100°C. Therefore, dry matter is estimated as a part of substance that does not evaporate at 100°C. The major difference in nutritional value on as fed basis is traceable to moisture content and dry matter. Calculation of relative cost of nutritional value involves consideration of moisture content.

Introduction

Pruning is the practise of cutting back, shaping, or removing branches, shoots, or leaves from plants in order to improve their overall health, regulate their development, make them seem nicer, or increase their output. Thinking about the plant’s requirements, growth habits, and intended results is essential in this horticulture discipline. Pruning is an essential tool in arboriculture, gardening, and agriculture; its use varies from plant to plant and from purpose to goal, from encouraging strong development to improving the plant’s aesthetic appeal. In the grand scheme of things, pruning is a deliberate action taken to improve the health and productivity of plants in their natural habitats.

Pruning is a regular part of plant maintenance involving the selective removal of specific plant parts. Although shoots and branches are the main targets for removal, roots, flower buds, fruits and seed pods may also be pruned. Pruning wounds plants, but plants respond differently to wounding than do animals. In plants, damaged areas are covered by callus tissue to close wounds. Another response to pruning occurs inside plants. Chemical boundaries form around wounded areas, walling off or compartmentalizing the wounds. Compartmentalization limits any decay that results from wounding, or from the natural death of branches. Use pruning techniques that minimize plant wounding and speed wound closure.

5.1 Pruning
One of the most crucial aspects of mulberry farming is pruning. It involves methodically cutting off some branches in order to give the mulberry plants a manageable size and form as well as to encourage the bud to sprout and develop new branches. The primary goal of pruning is to increase leaf output and quality. In addition, it is used to harvest the shoots for silkworm rearing and to do various inter-cultivation tasks including weeding, ploughing, applying manure and fertilizer and proper timing of the rearing of silkworms. To prune without breaking the bark or stem, use a sharp secateur, pruning saw or sickle. The plants in rainfed environments are only ever trimmed once a year, at a height of 10–20 cm above the ground level in June or July when the monsoon season begins. After that the plants are let to grow into bushes with many branches till the next year. Leaf harvesting is limited to the leaf picking method.
a) Pruning Types: Pruning is categorized according to the height of the stem at which it is cut. Bottom pruning is the practice of cutting the main stem at ground level after each harvest so as not to allow a stump to emerge. When low-cut pruning is used, the main stump of the plant is developed by cutting it 10 to 30 centimetres above the ground. At around 60 and 100 centimetres above the ground, medium and high cut pruning is performed respectively.

In recent years, pseudocereals and alternative cereal substitutes are gaining attention from agriculture, processing and consumer point of views. The 14 justification for such changes can be found in the recent thought of reextending the range of starch rich seeds available in human diet, because from world-wide perspective cereals consumption is largely restricted to the cereals wheat, rice and maize (Venskutonis and Kraujalis, 2013). In upcoming years, the increase in population can lead to an unfavorable pressure on agriculture and nutrition needs.

Introduction
World food security has become largely dependent only on a handful of crops like; wheat, rice, and corn which are the major cereals that support more than 50% of the global calorie demand. While these grains are an essential part of various diets, however they are devoid of essential micronutrients. As a result, it is estimated that micronutrient deficiency affects an estimated 2 billion people worldwide raising health concerns over our high dependence on cereal crops. As agriculture sector is considered as backbone of nation and therefore utilizing a handful of crops have placed the global food security at risk. In this context, Food and Agriculture Organization (FAO) has identified many plants as under-utilized, which can significantly contribute for improving nutrition and health, enhance food basket and livelihoods, future food security and sustainable development (FAOSTAT, 2023). These underutilized crops offer an immense potential in the functional food sector to combat hidden hunger crisis and offer the options of income generation.
Cereals such as rice, maize and wheat are monocots and belong to family Poaceae, however buckwheat, chia, quinoa and amaranthus although resembles with cereals are dicots and are categorized as Pseudocereals. These crops have been placed under the category of important crops by UNESCO due to dwindling cultivation and exploitation in the wild besides, these pseudocereals has gained a worldwide importance in the nutraceutical industry due to the rich nutritional profile compared to cereals and are considered as crops of the 21st century (Pirzadah et al., 2020). These are enriched with various active principals such as; polyphenols, flavonoids, amino acids, dietary fibre, lignans, vitamins, minerals, antioxidants, unsaturated fatty acids and other essential components like fagopyritols. The protein quality and quantity in pseudocereals is far better compared to cereals and this property qualifies their entry into the functional food industry (Rodriguez et al., 2020). Pseudocereals are rich in amino acids such as; arginine, tryptophan, lysine and histidine that proved essential for infant and child health thus, projected pseudocereals as an appropriate food supplement for child nutrition. The food production agro-systems must be adapted to climate change to ensure food security and stability. However, pseudocereals are climate resilient crops and can be grown even on marginal lands that are not fit for cereals thus, are considered as future crops to tackle mal-nutrition and future food crises. To explore these neglected or lost crops, there is an increasing interest in research and development that needs heightened direction and focus (Mabhaudhi et al., 2019). In this context, there is a need to adopt recent biotechnological interventions for the crop improvement and domestication of pseudocereal crops. This chapter focuses on nutritional profile of emerging pseudocereals viz., Buckwheat, Chia, Quinoa and Grain amaranth, phylogeny, genetic resources, breeding behavior and advanced breeding methods and their integration into the global food system