eChapters

ABSTRACT

This paper is an attempt to draw the attention of readers of this volume to understand the importance of documentation and preservation of earth’s resources.  

Introduction

Earth has innumerable treasures that were and are being exploited by mankind. Geo-resources in the form of minerals, non-renewable hydrocarbons, paleontologic relics and related evidences indicate earth’s evolutionary history. Continuum of biological activity albeit varying rates of proliferation had resulted in accumulation of natural resources embedded in Earth and distributed at specific geographical niches. Biological perspective of earth history is significant in unraveling bio-geo-sphere interactions through geological time. Fossil records of invertebrates, vertebrates and plants are signals of major changes in earth’s evolutionary history. These constitute unparalleled windows to understand continental separation. Intercontinental and intra continental migration of living forms are also traced through fossil study.

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.

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

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

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

To explain the main concepts and elements of a participatory curriculum development process

To present practical skills and knowledge leading to the effective design of teaching and learning

To build confidence of the learners in applying new methods and approaches to teaching and field extension work.

To demonstrate application of participatory approach in development of Extension curriculum

This Toolkit emphasizes an active learning approach, based on the process of experience, reflection and action. The key principle is that people do not learn simply by passive listening. They need to be involved from beginning of the training and designing of the program.

Quantitative Versus Qualitative Inheritance

There is a continuum of traits being inherited as a Mendelian trait with simple inheritance and traits having quantitative inheritance without well separated classes and with many genes involved.
Classification of traits in relation to mode of inheritance and environmental tolerance are shown in Figure 1.2. First there are the well known traits with simple Mendelian mode of inheritance. The trait with quantitative genetic inheritance is caused by segregation of many gene pairs, each with small effect. At the same time the trait is influenced by a lot of minor environmental effects.
Diseases will often be ‘either/or traits’ as the simple Mendelian traits. Cases in which the severity of the disease has a normal distribution can also be found. In many production diseases the disease only occurs when a genetically prone individual is exposed to adverse environmental effects. Figure 1. Classification  of traits in relation to mode of inheritance  and environmental tolerance. 
 

1. Introduction:- Overview of Good Laboratory Practices (GLP)
1.1 Definition and Scope of GLP
GLP refers to a set of standards that govern the organizational processes, personnel responsibilities, and conditions under which laboratory studies are conducted. It aims to regulate research environments and ensure the generation of high-quality, traceable, and reproducible results. The scope of GLP encompasses a wide range of scientific fields including pharmaceuticals, agrochemicals, veterinary products, and environmental safety assessments. Good Laboratory Practices (GLP) regulations were developed by the Organization for Economic Co-operation and Development (OECD) and well adopted by regulatory agencies world-wide. The primary objective of GLP is to promote high standards of accuracy and integrity in non-clinical research to protect human health and the environment.
1.2 Principles of GLP
The fundamental principles of GLP include:
• Study Planning and Protocol Development: Every study must have a clear plan and defined objectives, which should be outlined in a comprehensive protocol.
• Personnel Responsibilities: The roles of study directors, scientists, quality assurance personnel, and support staff are clearly defined.
• Test Systems and Facilities: Laboratories must maintain appropriate facilities and control systems for conducting studies. These include properly calibrated equipment, controlled environments, and standardized procedures.
• Data Integrity and Reporting: Data must be meticulously recorded, verified, and archived, ensuring it is traceable and reproducible.
• Study Documentation and Archiving: All records, raw data, and reports must be stored and accessible for a period defined by regulations, ensuring transparency and reproducibility.

Molecular diagnostics has witnessed groundbreaking technological advances in recent times, starting from automated sample preparation to real time amplification technology. Possibility to develop and run assays for ever increasing number of clinical queries has become effortless due to the technological advances. However, the state of the art in quality assurance and quality control practices for molecular diagnostics has seldom been up to the expectations if one considers accuracy for once in a lifetime genetic tests or new genetic test targets appearing almost every single day. In the face of such issues, clinical laboratories are struggling to develop appropriate quality assurance programs for the molecular diagnostic tests they conduct. In addition, legislation and government policies threaten to increase oversight of genetic tests to a level not seen before in laboratory medicine, for example, FDA’s guidance on in vitro diagnostic multivariate index assays (IVDMIA).

To relate patient results to previous test results or to absolute values in clinical practice guidelines, those results need to be comparable across time and methods. This may either be achieved by designing the identical value across methods and test versions or by using reliable and stable conversions. Therefore, the establishment of international standards and reference materials is of paramount importance. Monitoring molecular test system outputs and statistical analysis might provide a good way to obtain such standards; however, such traditional QC (Quality Control) strategies can be slow to take root in molecular diagnostics.This chapter highlights general and specific issues relevant for quality assurance and quality control in the routine molecular diagnostics laboratory and guidelines for designing quality manuals for a molecular diagnostic laboratory.