eChapters

Apoptosis is triggered by extrinsic and intrinsic stimuli as radiation, oxidative stress and genotoxic chemicals. It is defined by characteristic changes in the nucleus morphology including chromatin condensation and fragmentation, overall cell shrinkage/rounding and formation of apoptotic cell bodies. Among the many markers of apoptosis, DNA fragmentation is considered a hallmark. The DNA ladder assay described here is a simple, sensitive, cost-effective and rapid method for estimating apoptosis in cells.

Isolation of Genomic DNA

Genomic DNA from primary cells culture of PMNC and splenocytes of rats is isolated by using standard phenol-chloroform DNA isolation protocol of Sambrook and Russel (2001) as described below:

1. Preparation of primary cells culture of different cells, namely-PBMNCs, thymocytes and splenocytes form Wistar rats and viability checked by trypan blue dye exclusion test.

2. 106 cells/ml of primary cell culture is used and seeded in 24 well culture plates containing along with treatment with xenobiotics at different µM concentration.

3. Cell culture plates are incubated in CO2 incubator at 37°C with 5% CO2 for 12 hrs.

4. The cells are collected in 1.5 ml of micro centrifuge tube and centrifuged at 3200 rpm for 10 min at 4°C; supernatant is discarded and pellet is washed with PBS (pH 7.2).

5. DNA extraction buffer (500 µl/tube) is added in tubes. Gently tap to disperse the cell pellet in extraction buffer and keep in water bath for 1.0 hr at 37 °C (it is to ensure that pellet is completely suspended so that cells are accessible to SDS and Proteinase K).

6. 10 % SDS is added (20 µl/ml) to cell suspension and the tubes are gently mixed by inverting the tube. The contents of tube become viscous which indicates lysis of cells. After that sample is handled gently from onward to avoid shearing of DNA.

7. Proteinase K (15 µl) of 20 mg Proteinase K/ml of buffer is added in tubes in two pulse i.e. half the requirement is added to tube in 1st pulse, mixed gently end to end and kept in water bath at 50°C. After 3-4 hrs, the second pulse of the remaining amount of Proteinase K is added and tubes are incubated at 50°C overnight.

8. Equal volume of equilibrated phenol (Tris saturated phenol pH> 7.8 ) is added in the tubes next day morning. Mix the contents of tube gently by inverting the tube for 15 min till a light coffee coloured uniform solution without any balls of phenol is formed and then centrifuge at 3400 rpm for 15 min.

Burn wounds are considered to be one of the most devastating kinds of trauma that affect the physical, physiological and psychological well being of an individual. Based on the depth of the affected area, burn wound is divided into three categories: first degree (superficial), second degree (partial) and third degree (full thickness). Extensive and deep burns result in scar formation and subsequent deformation of the affected part. Healing of burn wound involves several interrelated and overlapping stages such as inflammatory, proliferative and remodeling phases. The interaction of a variety of mediators, cells and extracellular matrix proteins are involved in this three phases of healing.

Burn wound therapy has been performed in various ways such as bioengineered skin grafts and administration of growth factors. Small intestinal sub-mucosa (SIS) is a resorbable xenogenic bioscaffold that has induced constructive remodeling in variety of animal models. It is unique from other acellular matrices as it contains few growth factors such as fibroblast growth factor (FGF) and transforming growth factor (TGF) that play a role in the regenerative process. Chitosan is currently proposed to be one of the most promising polymers in wound dressing development and it accelerates wound healing. Epidermal growth factor is known to stimulate growth of keratinocytes in vitro, thus it might be expected to promote wound healing.

In this chapter efficacy of Chitosan and porcine acellular small intestinal sub-mucosal (SIS) matrix seeded with bone marrow derived mesenchymal stem cells (BM-MSCs) and recombinant murine epidermal growth factor (m-EGF) on healing of full thickness burn wound in rat model is described.

High-performance liquid chromatography or high-pressure liquid chromato graphy (HPLC) is a chromatographic method that is used to separate a mixture of compounds in analytical chemistry and biochemistry so as to identify, quantify or purify the individual components of the mixture. It involves the injection of a small volume of liquid sample into a tube packed with tiny particles (1.8- to 5-micron (μm) particle size called the stationary phase) where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.

Aflatoxins are toxic and carcinogenic compounds produced as metabolites by many Aspergillus species. This fungus invades habitats such as soil, grain, nuts, or decaying vegetation when- ever suitable conditions such as high moisture and temperature are met. B1 is the most toxic of at least 14 different types of naturally occurring aflatoxins. Therefore, many countries and regulatory agencies impose strict limits on B1 as well as other common aflatoxins such as B2, G1, and G2 despite exhibiting lower toxicity. The U.S. FDA set the action level to20 μg/kg (ppb) for human food and to 300 μg/kg (ppb) for animal feed. Aflatoxin B1 is extremely widespread and is present in food and feed products such as cottonseed, corn, and peanuts. It is highly toxic and has been classified as a group 1 carcinogen by the WHO. The aflatoxins B2 , G1 , and G2 are usually found accompanying B1 , in lower concentrations in the contaminated samples. The order of toxicity is AFB1 > AFG1 > AFB2> AFG2.

The cytological insult due to exposure of different environmental pollutant viz. pesticides, herbicides etc. impose great loss to the vitality and health of the individuals. The basic pathophysiological distrubances following the expo sure of these deterrents to the different visceral organs leads to dysfunction of these organs. Imbalance between body’s antioxidant defense and peroxidation of the lipid layer of cells’ causing free radical generation is defined as Oxidative stress. Free radicals are the molecules or entity that contains one or more than one unpaired electron. Pathophysiology of several diseases are associated with the generation of oxidative insult and many xenobiotics associated toxicity are also a result of oxidative damage and generation of reactive oxygen species and reactive nitrogen species. Alzheimer’s Disease is one of the fine example of oxidative damage associated neuronal degeneration.

Oxidative stress can be estimated using following parameters:

1. Lipid peroxidation (Paula et al., 2005)

2. Reduced glutathione (Sedlak and Lindsay, 1968)

3. Superoxide dismutase (Madesh and Balasubramanian, 1998)

4. Catalase (Aebi, 1983)

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.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using a large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of the drug discovery process can save time and money both by eliminating least beneficial NCEs.

Preparation of Reagents

Culture media

For the culture of hepatocytes medium M199 (GibcoBRL, USA) is used. One pouch of the dry media is dissolved in 1 liter of filtered distil water. The pH of the media is adjusted by adding 0.4% sodium bicarbonate to be around 7.4. The media is than sterilized by filtration through seitz filter (0.22 mm) and stored in 50 ml sterilized conical flasks.

Abstract

Patent infringement is the act of unauthorized making, using, offering for sale or selling any patented invention within the India. In case of patent infringement it is necessary for the patentee to compare the claims granted to him with the allegedly infringing product an check whether there is an infringement – whether the infringing product falls within the scope of the claims of the patent. In such a case the patentee may sue for relief in an appropriate court. The patentee may ask for permanent injunction and damages on account of the infringement. It is the crusade of the inventor asserting his rights against an infringer and the law recognizes the importance of this right; because, without such positive and assertive rights, industrial progress would be hard to come by

Abstract

Patent is a monopoly right which is granted by the Government to the applicant for the full disclosure of an ‘invention’ which is ‘new’, ‘inventive’ and ‘industrially applicable’. Thus, before filing a patent application, the inventor must be sure that the work done by him is new and inventive. Further, during the examination of the patent application, it is required to be verified that the invention claimed in the disclosure/specification is new, inventive and industrially applicable. Since the search for the ‘Novelty’ of the invention is carried worldwide, it becomes all the more important for the inventor and the examiners to search for the prior art thoroughly. Searching for the prior art (i.e. state of the art) across the globe in around millions of documents both patents and non-patent documents, is like ‘searching a needle in the haystack’. However, by using proper keywords and classification techniques, it is possible to arrive at the closest prior art available. Each patent application is classified by the Indian Patent Office and thus attributed to one or several fields of technology. The classification scheme applied is the International Patent Classification (IPC).

Abstract

Describes the genesis, organization and Internet representation of International Patent Classification (IPC). Provides the outline of seventh edition and useful URLs related to it.

Keywords: International Patent Classification, Patent Classification, Classification, Internet representation

Introduction

The International Patent Classification, commonly known as the IPC, is based on an international multi-lateral treaty administered by World Intellectual Property Organization (WIPO). This treaty is called the Strasbourg Agreement concerning the IPC, which was concluded in 1971 and came into force on October 7, 1975. The Agreement aimed to overcome the difficulties caused by using diverse national patent classification system. The common classification system defined by the Agreement is applied to patent and utility model documents. The Agreement is open to States party to the Paris Convention for the Protection of Industrial Property.

Livestock is an integral component of Indian agriculture which is primarily a mixed crop-livestock farming system and plays an important role in the socio- economic life of India. Livestock contributes significantly to the growth and development of agricultural sector by providing a rich source of high quality products such as milk, meat and eggs and by supporting livelihood of more than two third of rural population. Livestock has a crucial and rapidly increasing role infood security and economic stability worldwide Livestock sector contributes for 4.11% of GDP and 25.6% of total Agriculture GDP. The importance of livestock has been undergoing a steady transformation with the contribution of livestock in total agriculture and allied sector Gross Value Added increasing from 24.32% in 2014-15 to 28.63% in 2018-19. Owing to factors such as population and economic growth, urbanization, improvements in transportation and storage practices,its future importanceformeeting human nutritional requirements of food and fiberand a major contributor to agricultural GDP is evident.

To alleviate the concerns raised by a rapidly growing demand of food security and the need of ensuring sustainability of natural resources, we need to better understandthe ways which wouldspeed up commercialization of livestock production so that it can contribute effectively to the bio-economy. Livestock performance and productivity can be improved by adopting proper diet and feeding practices, improved farm management and disease controlstrategies.

Keeping in view the importance of intellectual property rights and their protection, Journal of Intellectual Property Rights (JIPR) was started in 1996. The objective of the journal is enhancing communication between policy makers, organizational agents, academics, and managers on the critical understanding and research on intellectual property; and to promote the development of the newly cultivated research field. The journal publishes contributed/invited articles, case studies and patent reviews; technical notes on current IPR issues; literature review; world literature on IPR; national and international IPR news, book reviews, and conference reports.

JIPR is covered in the abstracting/ indexing services namely Scopus, EBSCO host Electronic Journals Service, Directory of Open Access Journal, Library & Information Science Abstracts database and Legal information Institute of India database.

True/False

Q.1.    In many epidemiological studies with substantial follow up time, it is very often an interest to study the time taken for a particular event to occur.
Q.2.    The time taken for death or graft failure since transplant, time taken for relapse after treatment, time to death after treatment etc. are examples for survival time.

True/False

Q.1.    The accuracy of the measurement refers to how close the measured value is to the true value.
Q.2.    The precision refers to how close a set or group of measurements are together.
Q.3.    If the processes of measurement are repeated under identical condition, then precision explains that up to what extent these repeated values are reliable.

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.