Editorial Board- BioEvolution

Chief Editor Kanika Sharma, PhD

Professor Department of Botany, University College of Science,

M.L.S. University, Udaipur, Rajasthan, India

Managing Editor Arti Goel, PhD

Assistant Professor, Amity Institute of Microbial Biotechnology, Amity University, Sector- 125, Noida (U.P.)

Editors / Reviewers Nidhee Chaudhary, PhD

Professor Amity Institute of Biotechnology, Amity University Uttar Pradesh

Sector-125, Noida

Arpita Bhattacharya, PhD Associate Professor,

Amity Institute of Nanotechnology, Amity University, Sector- 125, Noida (U.P.)

Era Upadhyay, PhD Assistant Professor,

Ansal Institute of Technology & management, Sector C, Pocket 9, Sushant Golf City,

Lucknow (U.P.)

Prachi Bhargava, PhD Assistant Professor

Department of Biotechnology, Sri RamSwaroop Memorial University,

Deva Road, Lucknow (U.P.)

Rajshree Saxena, PhD Lecturer,

Amity Institute of Microbial Biotechnology, Amity University, Sector- 125,

Noida (U.P.)

Editorial

As an editor of BioEvolution I anticipate that this magazine would be of immense value and will be definitely useful to science and technology personnel in their thinking process. This online magazine will also offer a window for new perspectives and directions in each area of Microbiology, Immunology, Molecular Biology, Nanotechnology and Applied Sciences.

BioEvolution magazine is a periodical publication with news, opinions and reports about above mentioned areas for a non-expert audience. A periodical publication for scientific experts, in contrast, is called a "scientific journal". Science magazines are read by non-scientists and scientists who want accessible information on fields outside their specialization. Science presents us with a beautiful paradox: uncertainty as the optimal path toward certainty.

One of the best things about editing a magazine is that you learn about all kinds of people, places, and things you otherwise would not. I am very much grateful to GIAP Journals for giving me this golden opportunity to serve community through this magazine.

I also pay my sincere thanks to Chief Editor Dr Kanika Sharma for guidance, our reviewers, editors and technical team working behind the issue.

We pay special thanks to Dr Sachin Wazalwar, Assistant Professor, RCERT, Chandarpur, Maharashtra, India for wonderful and lively cover page for this issue.

Last but not least we are thankful to almighty for bestowing wisdom and peace upon us and putting serving attitude in our heart.

Arti Goel, PhD

Managing Editor

BioEvolution, GIAP Journals

Vol 1 (1), February 2014 ISBN 978-81-925781-5-6

Contents

Editorial Team

Editorial Message

1. CRYPTOCOCCOSIS: AN OPPORTUNISTIC SECONDARY INFECTION IN HIV PATIENT 01

Namita Bedi

2. GREEN NANOTECHNOLOGY 03

Arti Goel and Sneha Bhatnagar

3. MICROBES IN AGRICULTURE AND MEDICINE 05

Arti Goel

4. ARTIFICIAL ORGAN FORMATION WITH TISSUE ENGINEERING: A NOVEL

BIOMEDICAL APPROACH FOR SUSTAINING LIFE 09

Sadia Khan and Arti Goel

5. MOLECULAR DIAGNOSIS OF CANCER 12

Vartika Rai and Pranita Roy

6.RECTROACTIVE INHIBITION AND ITS EFFECT ON MEMORY 20

Ritu Rani, Ketki Sharma and Aparna Sarkar

7. ROLE OF PCR IN DIAGNOSTICS 23

Amit Kumar, Ritu Rani and Arti Goel

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CRYPTOCOCCOSIS: AN OPPORTUNISTIC SECONDARY INFECTION IN HIV PATIENT Namita Bedi

Assistant Professor, Amity Institute of Biotechnology,

Amity University, Noida, India

grovernamita@yahoo.co.in

An infection that occurs more frequently or is more severe in people with weakened immune system, such as people with HIV

or receiving chemotherapy , than in people with healthy system is called opportunistic infection. These infections are called

opportunistic because they take advantage of your weakened immune system and they can cause devasting illness. Most life

threatening opportunistic infection occur when your CD4 count is below 200 cells/mm3.Opportunistic infection are the most

common cause of death for people with Hiv/AIDS. There are many opportunistic infections related to HIV are tuberculosis,

cryptococcosis, syphilis, Listeria etc.

Among this Cryptococcus is a major opportunistic fungal pathogen in HIV pandemic countries worldwide. Cryptococcus is a

basidiomycetous yeast that survives environmentally in the sexual form, producing hyphae with terminal basidiospores (chains

of unbudded yeast). These basidiospores may break off and become aerosolized, and at 3 microns in size, are small enough to

deposit in the alveoli .In the majority of hosts, infection is asymptomatic. However, in the patient with severe cell-mediated

immunodeficiency, the organism may enter the circulation and survive in vivo in the haploid, asexual state leading to

disseminated disease. Characteristics of Cryptococcus that permit its survival within the host include a polysaccharide capsule,

which allows the organism to escape phagocytosis. Further, the phenol oxidase enzyme uses catecholamines as substrate to

produce melanin, which accumulates in the cell wall. It is the use of catecholamines that may provide a predilection for

involvement of the central nervous system (CNS).

CLINICAL MANIFESTATION OF CRYPTOCOCCUS

The etiological agent of Cryptococcosis differ in their virulence, geographic distribution, pathogenicity, clinical picture and the

therapeutic outcomes of infections. For example, Cryptococcus gattii typically causes a pulmonary infection in

immunocompetent hosts, but may also cause disease among HIV-infected persons. Pulmonary disease usually presents as an

acute pneumonia or as non-calcified granulomas, which are often difficult to detect radiographically. Some patients develop a

prolonged cough or dyspnea due to chronic pneumonia.

Cryptococcus grubii serotype A is recovered worldwide and accounts for over 90% of all cryptococcal infections and more

than 99% of cryptococcosis cases in AIDS patient. Cryptococcal meningitis remains an important cause of morbidity and

mortality in the AIDS population, especially in the developing world. Cryptococcal meningitis generally presents with a

headache of insidious onset and several weeks duration. Fevers usually do not occur until late in the disease course, and nuchal

rigidity is often absent.In AIDS patients with cryptococcal meningitis, extraneuronal involvement is quite common, as high as

50% in one case series. Common sites of infection include the lungs, bone marrow, skin, and genitourinary tract. Cutaneous

dissemination may be seen in about 10% of cases (typically described as molluscum-appearing skin lesions) and as osseous

involvement in approximately 5%.While cryptococcal meningitis generally occurs in immunocompromised patients, it can be

seen in immunocompetent individuals as well.

DIAGNOSIS OF CRYPTOCOCCUS The diagnosis of Cryptococcosis is most often made by the latex agglutination test for capsular polysaccharide antigen. This

antigen can be obtained from either cerebrospinal fluid (CSF) or serum, and when present in CSF, is over 90% sensitive and

specific for the diagnosis of cryptococcal meningitis. False positives can occur though, particularly in the presence of a positive

rheumatoid factor. In resource limited settings, the latex agglutination antigen test is often not available, thus methods of

detection of the organism include blood cultures CSF culture, and India ink smear. If isolated from culture, Cryptococcus

appears as singular, narrow-based budding yeast that is urease negative and can be distinguished by its preferential growth on

birdseed agar. The diagnosis of cryptococcal meningitis is established by lumbar puncture which usually shows elevated

opening pressure, high protein, and elevated white cell count. Indicators of more severe disease on CSF sampling include

elevated opening pressure, low glucose, leukocyte count less than 20 cells per mm3, elevated cryptococcal antigen titer, and

presence of the organism by India ink stain.

Recently, a new cryptococcal antigen (CRAG) detection assay has been developed by Immuno-Mycologics (CrAg LFA;

IMMY, Norman, OK). The lateral flow assay (LFA) is an immunochromatographic assay that functions as a dipstick test. Test

strips are coated with monoclonal antibodies to detect cryptococcal antigen; the presence of two bands (control and test) in the

test zone indicate a positive result, whereas a single band (control) indicates a negative result. This dipstick test is stable at

room temperature, requires minimal laboratory infrastructure and technical skill, and provides rapid results. Data indicate that

sensitivity in serum and CSF is comparable to that of LA and EIA. Use with urine and whole blood is currently being

evaluated. As of August 2011, the assay has received the CE mark of approval in Europe and US Food and Drug

Administration approval for use in serum.

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MOLECULAR APPROACH

Several molecular typing methods, including PCR-fingerprinting, randomly amplified polymorphic DNA (RAPD), PCR-

restriction fragment length polymorphism (PCR-RFLP), amplified fragment length polymorphism (AFLP), microsatellite

typing, multilocus microsatellite typing (MLMT) and multilocus sequence typing (MLST), have been developed for the

investigation of the epidemiology of species belonging to the C. neoformans/C. gattii species complex . MLST is a typing

system that has several advantages over other commonly used typing methods, because the technique is highly reproducible

and MLST sequence data can be stored in internet database. Thus, the data are portable and exchangeable between laboratories.

Recently, seven unlinked genetic loci, i.e.CAP59, GPD1, IGS1, LAC1, PLB1, SOD1 and URA5, that represent housekeeping

genes, virulence factor coding genes and the intergenic spacer of the ribosomal DNA have been selected for MLST analysis of

the C. neoformans/C.gattii complex by the International Society of Human and Animal Mycoses (ISHAM) working group on

“Genotyping of C. neoformans and C. gattii”.

Previous studies that used MLST and AFLP to investigate the population structure of C. neoformans var. grubii showed a

correlation between both methods and grouped the isolates into three genetically different subgroups, named AFLP1/VNI,

AFLP1A/VNII/VNB and AFLP1B/VNII . The AFLP1/VNI and AFLP1B/VNII genotypes occur globally and form a

monophyletic cluster, whereas the AFLP1A/VNB genotype

INTEGRATED APPROACH TO THERAPY OF CRYPTOCOCCUS AND HIV

In areas where cryptococcal disease prevalence is high, an integrated approach can help reduce early mortality among

HIV/AIDS patients starting ART. The recommended initial management of cryptococcal meningitis in patients with HIV

consists of the rapidly fungicidal regimen of amphotericin B (0.7 to 1 mg/kg/day) plus flucytosine (100 mg/kg/day). In the

HIV-positive patient with meningitis, lifelong maintenance therapy is often necessary. Following induction and consolidation

therapy lifelong suppressive treatment with fluconazole (200 mg daily) is recommended. If immune reconstitution occurs due

to initiation of a successful HAART regimen, the CD4+ T lymphocyte count remains greater than 100 cells/mm3 for at least

three months and the HIV viral load is low/undetectable, consideration of discontinuation of therapy can be made.Of note, a

minimum of 12 months of antifungal therapy should be administered before discontinuation.

In summary, worldwide, approximately 1 million new cases of cryptococcal meningitis occur each year, resulting in 625,000

deaths. Most cases of Cryptococcosis that occur among people with HIV/AIDS. Although the widespread availability of

antiretroviral therapy (ART) in developed countries has helped reduce cryptococcal infections in these areas, it is still a major

problem in developing countries where access to healthcare is limited. To prevent the spread of cryptococcal meningitis in HIV

patients “targeted screening” of Cryptococcal antigen should be done. A Cryptococcal antigen acts as a chemical marker for

infection and detected in the body weeks to months before the onset of symptoms. In this way cryptococcal meningitis can be

prevented in Hiv patient. The awareness about Cryptococcal meningitis will definitely help to reduce the mortality in HIV

patients.

REFERENCES

1. Ellis DH, Pfeiffer TJ. Natural habitat of Cryptococcus neoformans var. gattii. J Clin Microbiol. 1990;28:1642–1644.

2. Satischandra P, Mathew T, Gadre G, Nagarathna S, Chandramukhi A, Mahadevan A, Shankar SK. Cryptococcal

meningitis: Clinical, diagnostic and therapeutic overviews.Neurol India. 2007;55:226–232.

3. Centers for Disease Control and Prevention. 1993 Revised system for HIV infection and expanded surveillance case

definition for AIDS among adolescents and adults. MMWR.1992;41(R-17)

4. Kaplan JE, Benson C, Holmes KH, Brooks JT, Pau A, Masur H. Guidelines for prevention and treatment of

opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes

of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. Centers for Disease

Control and Prevention (CDC); National Institutes of Health; HIV Medicine Association of the Infectious Diseases

Society of America. MMWR. 2009. Apr, pp. 48–49.

5. Lotholary O, Poizat G, Zeller V, Neuville S, Boibieux A, Alvarez M, et al. Long-term outcome of AIDS-associated

cryptococcosis in the era of combination antiretroviral therapy. AIDS. 2006;20:2183–2191.

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GREEN NANOTECHNOLOGY Arti Goel

1* and Sneha Bhatnagar

2

1*Assistant Professor,

2M.Sc. Biotechnology Student

Amity Institute of Microbial Biotechnology, Amity University, Noida, India

*agoel2@amity.edu, peena_agrawal@yahoo.co.in

Green Nanotechnology is application of nanotechnology that envisions sustainability. Green nanotechnology is the result of the

worlds‟ fascination with tiny molecules being aware of the endless potential that this field has. Green nanotechnology is a great

industrial revolution. Green nanotechnology is supporting the development of sustainable solutions to address global issues.

Issues like energy shortages and scarcity of clean water, and many other areas of environmental concern, and being present in

environmentally-sustainable manufacturing processes can be resolved by green nanotechnology.

Green nanotechnology is used to develop clean technologies in order to minimize human health and potential environmental

risks. It is basically all about making green nanotechnology products and using these as a support to sustainability without

compromising human health.

Green Nanotechnology uses Green Chemistry, Green Engineering, and Industrial Ecology to discover the teeny-tiny magical

world of nanomaterials and nano-products that are without toxic ingredients, produced at low temperatures using less energy

and renewable inputs. Green nanotechnology miniaturizes products, which uses less material, are lighter to transport, and

thereby save energy and fuel.Green nanotechnology can have many applications, innovations and roles that can be developed

by its different products and can be of an enabling nature.

It can be used further for product developments and as a hike to today‟s technologies, for example:

a. Nanotechnology can play a role to a product to functionality (e.g. nanotechnology-enabled batteries)

b. Nanotechnology can enable green development and manufacturing processes without the final product containing any

nanomaterials

c. Applications of Green Nanotechnology like sensors, treatment of pollutants, green energy (for example, nano-enabled, fuel

cells), and green manufacturing.

GOALS OF GREEN NANOTECHNOLOGY:

The main goals of green nanotechnology is to produce nanomaterials that do not harm the environment and are eco friendly

also to derive specific nanoproducts from these nonmaterials and using them in human welfare.

Green nanotechnology works on the principles of “green engineering” which is designing of such products that conserve

natural resources and do not harm environment at the same time. It emphasizes the use of “sustainable chemistry” in order to

develop products that minimize the use of hazardous materials.

For making these materials and products the main considerations are making least use of toxic compounds with least amount of

energy being consumed. Also the materials should be renewable as long as it is possible.

ASPECTS OF GREEN NANOTECHNOLOGY:

The products of green nanotechnology are used to prevent harm from hazardous pollutants. They are widely used for

remediation of hazardous waste sites also to clean and desalinate polluted water. They also enable sophisticated sensing

devices to detect hazardous pollutants, plant pathogens and other toxins.

Wide range of products have been developed that play role in generation of energy. These are products like fuel cells,

thermoelectric devices, solar cells and improved batteries. All such materials are small sized and use less material thus in turn

save energy and fuel.

Another main aspect of green nanotechnology is human welfare. As these nanomaterials are produced with least possible harm

to the environment and release low amount of toxic and waste by products thus they do not generate any pollutants.

PRINCIPLES:

The main principles of green nanotechnology include-

a. Prevention of generation of wastes to a great extend as it is better to prevent waste than to treat or clean up waste after it is

formed.

b. Atom economy that is the conversion efficiency of a chemical process in terms of all atoms involved.

c. Less hazardous product synthesis should be there.

d. Safer chemicals and safer solvents also chemical products should be designed to preserve efficacy of function while

reducing toxicity.

e. Reduce derivatives - Unnecessary derivatization (blocking group, protection/ deprotection, temporary modification) should

be avoided whenever possible.

APPLICATIONS:

Applications of green nanotechnology products can be direct or indirect. These can be applied for-

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Desalination of water which is the removal of salts and minerals from water. One of the most promising applications for carbon

nanotube membranes is sea water desalination. These membranes will someday be able to replace conventional membranes

and greatly reduce energy use for desalination.

To sense and monitor environmental pollutants and to treat these pollutants and hazardous waste sites.

Other indirect applications may involve production of low weight nanocomposites for use in automobiles. From tyres to valves,

bearings, cylinders etc the use of green nanotechnology in automobile has made a remark.

To save fuels and fossil fuels and to in turn reduce pollution and also to reduce the use of many cleaning chemicals and

materials used for production processes.

INNOVATIONS IN GREEN NANOTECHNOLOGY-

Many innovations have been made in the field of green nanotechnology which has proved to be of great importance for the

future. Green nanotechnology and its innovations will result in a great impact to the economy from automobile to food. Some

of the well developed innovations are-

Nanotechnology for greener cars- A car is a complex combination of various components that can be converted to a greener

vehicle in various ways. These may include invention of non fossil fuel energy for these vehicles (ex- hydrogen fuel cells),

reduction in the consumption of fossil fuels by using nanocomposites, for tyre innovation as nanomaterials combined with

rubber.

Micro and nanofibrillar cellulose- Cellulose is a biodegradable material that has been extensively used in paper production.

Production of nanofibrils by wood pulp may lead to a great innovation for paper and packaging industry.

Efficiency in electronic components- Nanotechnology has made low energy consumption per bit possible along with high

performance in many electronic products. New „zero-power‟ systems are being developed that use minimal energy and convert

it to electrical energy.

Carbon nanotubes for green innovation- The basic requirement now a days carbon nanotubes is for transport and electronic

applications. Carbon nanotubes are of high strength, low weight and low density. Thus they can be used in various means of

transport as they have great potentials. On the other hand the use of carbon nanotubes for electronic purposes is much

beneficial due to its outstanding electrical conductivity and mechanical properties. They are much cost effective and

application specific.

CONCERNS RELATED TO INNOVATION OF GREEN NANOTECHNOLOGY:

While green nanotechnology brings products that reduce pollutants and are environment friendly major limitations here are the

cost and potential risks associated with the development. Advances have been made in the development of green

nanotechnology but it always has a risk of level of sustainability of certain green applications of nanotechnology.

The products of green nanotechnology might save energy and reduce carbon emission but the main concern also lies in the

energy usage for the upstream processing of these products. However, with ongoing research work that is being done in this

field it will surely result into a better future .

REFERENCES-

1. ACS Green chemistry institute and the Oregon nanoscience and microtechnology institute (2011) Green nanotechnology

challenges and opportunities, white paper. http://www.onami.us/PDFs/nano-whitepaper.pdf

2. Agboola, A.E.,Pike, R.W.,Hertwig, T.A., and Lou, H.H (2007). Conceptual design of carbon nanotubules process. Clean

nanotechnologies and environmental policies , vol 9, issue 4 , pp.-289-311.

3. OCED(2012a) , “ Transition to green innovation and technology ” , OCED science, technology and industry outlook 2012,

OCED publishing.

4. OECD (2013),”Nanotechnology for Green Innovation”, OECD science, Technology and Industry Policy Paper

,No.5,OECDpublishing.http://www.oecd-ilibrary.org/science-and-technology/nanotechnology-for-green-

innovation_5k450q9j8p8q-en

5. Nanotechnology for green innovation - a new OECD paper http://www.nanowerk.com/news2/newsid=30937.php

6. Research on Nanotechnology Applications: Green Nanotechnology for Past, Present, and Preventing Future Problems,

Barbara Karn, PhD Georgetown University and US EPAhttp://www.empa.ch/plugin/template/empa/*/70325/---/l=2

7. Green Nanotechnology: Straddling Promise and Uncertainty Barbara P. Karn and Lynn L. Bergeson

8. http://www.lawbc.com/uploads/docs/00051031.pdf

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MICROBES IN AGRICULTURE AND MEDICINE Arti Goel

Assistant Professor, Amity Institute of Microbial Biotechnology

Amity University, Noida, India

agoel2@amity.edu, peena_agrawal@yahoo.co.in

Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range

of shapes, ranging from spheres to rods and spirals. Bacteria are present in most habitats on Earth, growing in soil, acidic hot

springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and

animals, providing outstanding examples of mutualism in the digestive tracts of humans, termites and cockroaches. There are

typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water; in all, there are

approximately five nonillion (5×1030

) bacteria on Earth, forming a biomass that exceeds that of all plants and animals. Bacteria

are vital in recycling nutrients, with many steps in nutrient cycles depending on these organisms, such as the fixation of

nitrogen from the atmosphere and putrefaction. In the biological communities surrounding hydrothermal vents and cold seeps,

bacteria provide the nutrients needed to sustain life by converting dissolved compounds such as hydrogen sulphide and

methane. Most bacteria have not been characterised, and only about half of the phyla of bacteria have species that can be grown

in the laboratory. The study of bacteria is known as bacteriology, a branch of microbiology.

There are approximately ten times as many bacterial cells in the human flora as there are human cells in the body, with large

numbers of bacteria on the skin and as gut flora. The vast majority of the bacteria in the body are rendered harmless by the

protective effects of the immune system, and a few are beneficial. However, a few species of bacteria are pathogenic and cause

infectious diseases, including cholera, syphilis, anthrax, leprosy, and bubonic plague. The most common fatal bacterial diseases

are respiratory infections, with tuberculosis alone killing about 2 million people a year, mostly in sub-Saharan Africa. In

developed countries, antibiotics are used to treat bacterial infections and in agriculture, so antibiotic resistance is becoming

common. In industry, bacteria are important in sewage treatment and the breakdown of oil spills, the production of cheese and

yogurt through fermentation, the recovery of gold, palladium, copper and other metals in the mining sector, as well as in

biotechnology, and the manufacture of antibiotics and other chemicals.

ROLE OF BACTERIA IN AGRICULTURE:

The activities of bacteria are very important in agriculture in the following aspects:

(a) Decaying of organic substance: Most of the bacteria are very useful in bringing about decomposition of dead organic matter of plants and animals by the

secretion of enzymes. The enzymes convert the fats, carbohydrates and nitrogenous compounds into simpler forms, such as,

CO2 water, ammonia, hydrogen sulphide, phosphates, nitrates etc that are used as raw material by the green plants. Thus, these

bacteria not only decompose the organic compounds but also remove the harmful waste from the earth and thus function as

nature's scavengers.

(b) Fertility of the soil: Some bacteria maintain and others increase the fertility of the soil. They bring about physical and chemical changes in the soil

by converting insoluble materials into soluble ones. These bacteria are the ammonifying, nitrifying and the nitrogen fixing

Bacteria.

(c) Ammonifying Bacteria: The decay bacteria decompose the proteinaceous compounds into amino acids, which are reduced to ammonia by ammonifying

bacteria. The free ammonia combines in the soil to form ammonium salts. This conversion is known as ammonification.

Examples are Bacillus ramosus, B. vulgaris etc.

(d) Nitrifying Bacteria: These bacteria convert ammonium salts into nitrates, which are absorbed by the plants. The nitrifying bacteria are the

Nitrobacter and Nitrosomonas. The Nitrosomonas oxidize the ammonium salts into nitrous acid, which forms nitrites in the

soil. The Nitrobacter then converts the nitrites into nitrates. This conversion of ammonium salts into available nitrates is called

nitrification.

(e) Nitrogen Fixing Bacteria: These bacteria take up nitrogen from the atmosphere and convert it into organic nitrogen compounds. It is known as nitrogen

fixation. The nitrogen-fixing bacteria are of two types. One type includes Azotobacter and Clostridium, which live freely in the

soil and fix nitrogen of the air in their bodies in the form of nitrogenous organic compounds. The other types of bacteria are the

nodule bacteria, the Bacillus radicicola. Rhizobium lives as symbiont in the roots of leguminous plants and forms nodules.

These bacteria absorb free nitrogen from the bacterial cell. The leguminous plants thus enrich the fertility of the soil. They are

grown for green manuring and rotation of crops.

ROLE OF BACTERIA IN INDUSTRY AND MEDICINE:

There are many bacteria’s that are important for medical uses. Some have even been used for bacteriological warfare.

However, for a bacterium to be of use in medicine, it need to have some sort of property we want. A bacteria is normally

dangerous for humans when it rapidly multiply and produces toxins. In order to produce toxins and multiply it eats away on us.

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Some of the toxins a bacteria produces can actually be of help and the specific bacteria’s for that are cultivated in a nourishing

mixture and the toxins are distilled out. These toxins can be put in medicine afterwards and can help to cure various illnesses,

even kill off other germs we don't want in our systems.

Bacteria also play a very important role in various industries. The products obtained as a result of bacterial activities cannot be

chemically prepared. Their activities are involved in the following industries:

(a) Preparation of Alcohols: Ethyl alcohol and butyl alcohol are manufactured by the bacterial activities in the sugar solution, e.g., Clostridium

acetobutylicum.

(b) Preparation of Vinegar: Vinegar is prepared by the activities of Acetobacter acetii in the sugarcane juice.

(c) Preparation of Butter, Cheese etc.: The preparation of butter, cheese etc. is done by bacteria. The Lactobacillus lactis is responsible for souring of milk resulting

in curd (dahi) preparation. Bacterial activities also impart the typical flavours.

(d) Preparation of Tea, Coffee etc.: Bacteria are very useful in preparation and flavouring of tea, coffee, cocoa etc. e.g., Micrococcus.

(e) Preparation of Tobacco: Tobacco leaves are cured and flavoured by the bacteria. Typical types of bacteria are cultured for this purpose, e.g.,

Micrococcus.

(f) Preparation of Hemp fibres: Fibres from the hemp are isolated after rotting the stems by activity of bacteria (e.g., Clostridium butyricum). The bacteria eat

up the protoplasmic tissues but leave the sclerenchyma fibres.

(g) Preparation of Leather and Tanning: The hairs and fats are removed from the skin by the action of bacteria in the leather industry.

(h) Preparation of Antibiotics: The bacteria are also used in the preparation of antibiotics. According to Sir Alexander Flemming, the growth of harmful

Staphylococcus is checked by Penicillium notatum. With this discovery, large number of antibiotics has been prepared which

are of great importance in the medical world. Tyrothricin, Subtillin, Polyximin-B, Bacitracin, Streptomycin, Aureomycin,

Terramycin are some well-known antibiotics.

Fungi are simple plant forms, and include mushrooms, molds, yeasts and mildews. Unlike other plants, however, fungi do not

have chlorophyll and are not capable of photosynthesis. Fungi have important culinary, medical, agricultural and industrial

uses. Fungi can be used to create dyes, medications and eco-friendly building materials.

Fungi are extremely useful organisms in biotechnology. Fungi construct unique complex molecules using established

metabolic pathways. Different taxa produce sets of related molecules, each with slightly different final products. Metabolites

formed along the metabolic pathway may also be biologically active. In addition, the final compounds are often released into

the environment. Manipulation of the genome, and environmental conditions during formation of compounds, enable the

optimization of product formation.

ROLE OF FUNGI IN AGRICULTURE

India is a country, mostly dependent on agriculture. Fungi play both positive and negative roles in agriculture. The harmful

activities are more than the helpful activities. Some of the saprophytic fungi in the soil decompose the dead material of animals

and plants. The enzymes secreated by these fungi convert the fats, carbohydrates and nitrogen compounds of the dead animals

and plants into simpler compounds such as carbon dioxide, ammonia, hydrogen sulphide, water and some other nutrients in a

form available to green plants. Some of them will be in the soil to form humus and the remaining go into air where they can be

used up as raw material for food synthesis. By liberating carbon dioxide these fungi participate in maintaining the never ending

cycle of carbon in nature. The carbon dioxide is very important for green plants in the preparation of food materials by

photosynthesis.

Some fungi are in symbiotic association with the roots of certain plants. Satisfactory growth of the plant can be observed only

when the specific fungal partner is present inside the roots of the plants. This type of association of a fungus and plant is called

mycorrhiza. Some nematodes are known to cause severe losses to agricultural crops directly and some transmit certain disease

causing viruses also. A few fungi (e.g., Dacty/aria) are known to destory the nematodes. These predatory fungi produce

mycelial loops. When the nematodes pass through, these loops get tightened up to catch the nematodes.

Similar to PGPR (plant growth promoting rhizobacteria), some rhizosphere fungi able to promote plant growth upon root

colonization are functionally designated as 'plant- growth-promoting-fungi’ (PGPF) .PGPF belong to genera Penicillium,

Trichoderma, Fusarium and Phoma. Several species of PGPF have been shown to trigger systemic resistance against various

pathogens in cucumber plants5. Plant growth promoting fungi (PGPF), which are non-pathogenic soil inhabiting saprophytes,

have been reported to be beneficial to several crop plants not only by promoting their growth but also by protecting them from

diseases. Among these PGPF, some isolates of Phoma sp. and Penicillium simplicissimum GP17-2 were highly effective in

inducing systemic resistance against cucumber anthracnose caused by Colletotrichum orbiculare. One of the primary functions

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of filamentous fungi in soil is to degrade organic matter and help in soil aggregation. Besides this property, certain species of

Alternaria, Aspergillus, Cladosporium, Dematium, Gliocladium, Helminthosporium, Humicola and Metarhizium produce

substances similar to humic substances in soil and hence may be important in the maintenance of soil organic matter.

ROLE OF FUNGI IN MEDICINE Fungi with antimicrobial and other biological activities can produce wide ranges of natural products which is why they are

used in drug manufacturing industries. They are widely used for the production of antibiotics, anti-cancer, vitamins and

cholesterol lowering drugs. Antibiotics produced by fungi, have gained a high popularity because of their extensive use in

disease treatment. The production of antibiotics by fungi was first discovered by Alexander Fleming in 1929. He discovered

Penicillin, the wonder antibiotic, which is produced by Penicillium notatum. Penicillin kills several bacteria and it has no

harmful effects on the human calls.

The limited use of penicillin against the vast number of diseases made the researchers to search for other antibiotics. This

search resulted in the discovery of several other antibiotics. Fumigallin from Aspegillus fumigatus inhibits certain phages and

amoebae. Griseofulvin, another antibiotic from Penicillium griseofulvm, is used against the skin diseases such as ring worm

and athletes foot. This antibiotic interferes with the wall formation of the disease causing fungi. Consequently the pathogens

cease to grow. The compound accumulates in skin and hair when taken orally and so it is effective against skin diseases. A

mixture of Alkaloids from Claviceps purpurea (causal organism of ergot of rye) is highly poisonous. This is used to control

bleeding during child birth. Clavacin, a substance extracted from Clavatia, prevents stomach tumors.

Fungi contain a number of compounds that can stimulate immune function and inhibit tumor growth in humans. Among these

compounds, those termed polysaccharides, which are large, complex chains of molecules constructed of smaller unites of sugar

molecules, are also found in lichens (a symbiosis of fungus and green alga), bacteria, and even from the cell walls of yeast (a

carbohydrate called zymosan). These immune-activating polysaccharides are similar to those found in more complex plants

such as echinacea and astragalas (a widely used Chinese herb) and they are now understood to do an amazing thing in our

bodies.

These giant polysaccharide molecules are similar to ones found in the cellular membranes of bacteria, and thus, trick our

immune system into believing it is being invaded, and accordingly, it mounts an immune response. While this perceived threat

poses no actual danger to our bodies, this immune response triggers the increase of a number of powerful immune activities

including macrophage and “killer” T-cell (white blood cell) activity.

But polysaccharides are not the only active components found in fungi, nor are immune and anti-tumor activity the only

influence they have. Smaller compounds such as terpenes and steroids present have also been shown to resist the growth of

tumors, and a number of what are called “protein-bound” polysaccharides have even shown to have antibiotic and antiviral

properties, as well as the ability to lower blood pressure and reduce blood-level lipids (fats) and sugar. These properties make

fungi especially useful in treating infections, flu, diabetes, various heart conditions, and according to many studies, perhaps the

HIV.

SUGGESTED READINGS:

1. Fredrickson J.K., Zachara, J.M., Balkwill, D.L. et al 2004. "Geomicrobiology of high-level nuclear waste-contaminated

vadose sediments at the Hanford site, Washington state". Applied and Environmental Microbiology 70 (7): 4230–41.

2. Higa, Teruo, Parr, J. 1994 (PDF). Beneficial and Effective Microorganisms for a Sustainable Agriculture and

Environment. Atami, Japan: International Nature Farming Research Center. pp.7.

3. Hogan, C.M. 2010. Bacteria. Encyclopedia of Earth. eds. Sidney Draggan and C.J.Cleveland, National Council for

Science and the Environment, Washington DC

4. Ishige, T., Honda, K., Shimizu, S. 2005. "Whole organism biocatalysis". Current Opinion in Chemical Biology, 9 (2):

174–80.

5. Rappé, M.S., Giovannoni S.J. 2003. "The uncultured microbial majority". Annual Review of Microbiology, 57: 369–94.

6. Sears, C.L. 2005. "A dynamic partnership: celebrating our gut flora". Anaerobe, 11 (5): 247–51.

7. Whitman W.B., Coleman D.C., Wiebe, W.J. 1998. "Prokaryotes: the unseen majority". Proceedings of the National

Academy of Sciences of the United States of America 95 (12): 6578–83.

8. Xu, Hui-Lian, 2001. Effects of a Microbial Inoculant and Organic Fertilizers on the Growth, Photosynthesis and Yield of

Sweet Corn. Journal of Crop Production, 3(1): 183-214.

9. Xu, H.L.; Wang, R; Mridha, A.U. 2001. Effects of Organic Fertilizers and a Microbial Inoculant on Leaf Photosynthesis

and Fruit Yield and Quality of Tomato Plants. Journal of Crop Production, 3(1): 173-182.

10. Yamada, K and Xu, H. 2001. Properties and Applications of an Organic Fertilizer Inoculanted with Effective

Microorganisms. Journal of Crop Production, 3(1): 255-268.

11. Yan, P.S. and Xu, H.L. 2002. Influence of EM Bokashi on Nodulation, Physiological Characteristics and Yield of Peanuts

in Nature Farming Fields. Journal of Sustainable Agriculture, 19: 105-112.

12. Anke, T. and Thines, E. 2007. Fungal metabolites as lead structures for agriculture. In: Exploitation of Fungi. Eds: Robson

GD, van West P, Gadd GM. CUP.

13. Suryanarayanan, T.S. et al. 2009. Fungal endophytes and bioprospecting. Fungal Biology Reviews, 23: 9-19.

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14. Hyakumachi, M. 1994. Plant growth promoting fungi from Turfgrass rhizosphere with potential for disease suppression.

Soil Microorganisms, 44s: 53-68.

15. Shoresh, M., Yedida, I. and Chat, I. 2005. Involvement of jasmonic acid/ethylene signaling pathway in the systemic

resistance induced in cucumber by Trichoderma asperellum T203. Phytopathology, 95:76-84.

16. Shivanna, M.B., Meera, M.S. and Hyakumachi, M. 1994. Sterile fungi from Zoysiagrass

17. rhizosphere as plant growth promoters in spring wheat. Canadian Journal of Microbiology, 40: 637-644.

18. Shivanna, M.B., Meera, M.S. and Hyakumachi, M. 1996. Role of root colonization ability of plant growth promoting fungi

in the suppression of take-all and common root rot of wheat. Crop Protection, 15: 497-504.

19. Meera, M.S., Shivanna, M.B., Kageyama, K and Hyakumachi, M. 1994. Plant growth promoting fungi from Zoysiagrass

rhizosphere as potential inducers of systemic resistance in cucumbers. Phytopathology, 84: 1399-1406.

20. Koike, N., Hyakumachi, M., Kageyama, K., Tsuyumu, S. and Doke, N. 2001. Induction of systemic resistance in

cucumber against several diseases by plant growth promoting fungi: lignification and superoxide generation. European

Journal of Plant Pathology, 107: 523-533.

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Authors will be given publication certificate and 20% royalty.

These projects / dissertations will be published in ISBN books and will be available through various websites and portals as reference material for next generation.

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ARTIFICIAL ORGAN FORMATION WITH TISSUE ENGINEERING: A NOVEL

BIOMEDICAL APPROACH FOR SUSTAINING LIFE Sadia Khan

1 and Arti Goel

2*

1 Student of M.Sc. Medical Physiology, Amity Institute Of Physiology and Allied Sciences

,

2*Assistant Professor, Amity Institute of Microbial Biotechnology,

Amity University, Noida, India

sadiakhan315@gmail.com, agoel2@amity.edu, peena_agrawal@yahoo.co.in

Tissue engineering is an emerging interdisciplinary field of applied biology and biomedical engineering which majorly

merges three different areas of study viz. biology, medicine, and engineering. It has a much broader aspects in biomedical

research than what we are considering here in this article and hence it has a multi-disciplinary approach of studies.

One of the most innovative approaches of this technique is artificial organ formation which mainly aims at organ

transplantation, restoring, maintaining or enhancing tissue and organ function. Besides these therapeutic aspects, it has

diagnostic applications as well where in-vitro organ formation is used to test drug metabolism and uptake, their toxicity and

pathogenicity (drug delivery).

The basis of this novel research technology lies in the fact that cells can be cultured in laboratory in the presence of

biomolecules and biomaterials using engineering and biomechanical aspects of design. Cells that are cultured in-vitro are

divided on the basis of their source. For instance, autologus cells are obtained from the same individual to which it has to

be re-implanted. Similarly, allogenic cells come from the donor of the same species whereas xenogenic cells come from

the donor of other species. Syngenic or isogenic cells, on the other hand, are derived from genetically identical organisms

such as twins or clones. Another category differentiates cell into primary i.e. donated from an organism; or secondary i.e.

donated from the cell bank. The pillar of this technique of artificial organ formation, however, rests on the ability of the

stem cells to divide and differentiate in culture to form different specialized cells. Stem cells are undifferentiated cells

which are divided into two categories:

a. Adult stem cells: which are multipotent i.e. they have the ability to differentiate into multiple types of cells.

b. Embryonic stem cells: which are pluripotent i.e. they have ability to differentiate into less types of cells. In the early

stages, they are totipotent i.e. only a single cell can differentiate to form all the specific cells of that organism.

Tissue engineering exploits these potentials of a stem cell to form tissues artificially in the laboratory and holds a promising

solution to overcome the problem of shortage of organ donors. Tissue engineering technique is solely based on the

biomaterials which are responsible for the scaffoldings: an artificial structure responsible for supporting the 3D tissue

formation. The biomaterials used for scaffolds must have some basic properties like biocompatibility and biodegradability;

high porosity for diffusion of vital nutrients, mechanically strong enough to be handled while surgery; its architecture

should match with the site/organ where it is to be implanted; and so forth. Besides scaffoldings, other regulatory factors

work together to design an artificial organ successfully. Three considerable factors that essentially lie behind the procedure

of organ formation are biomaterials, living cells, and bioactive molecules.

Biomaterials, as defined by the European Society for Biomaterials

(ESB), are ‘materials intended to interface with biological systems to

evaluate, treat, augment or replace any tissue, organ or function of the

body’. These biomaterials, as mentioned earlier, serve as a template for

regeneration in tissue engineering by forming scaffolds. Biomaterials

used for fabrication of scaffolds may be ceramics, synthetic polymers or

natural polymers. Ceramic scaffolds, like hydroxyapatite and tricalcium

phosphate, are highly stiff, less elastic and have a brittle surface. They

are fit for artificial bone formation. Synthetic scaffolds include

polystyrene and polyglycolic acid etc. Natural scaffolds comprise of

collagen, various proteoglycans, alginate-based substrates and chitosan.

Natural scaffolds promote excellent cell adhesion and cell growth. They

are also biodegradable and can be replaced by extracellular matrix.

Bioactive molecules provide microenvironment for the growth, differentiation and proliferation as well as regulation of the

tissue. This means that organ level tissue engineering is directed by active biomolecules by the means of signalling cues

directing morphogenesis and cell differentiation into tissue specific structures. Growth factors play essential role tissue

regeneration and are expressed during different phases of tissue development. Organ-level morphogenesis also requires the

presence of tissue-specific signals, specifically growth factors to induce the formation of new tissue. Thus, the

microenvironment that surrounds a construct comprises of required substrates along with the bioactive molecules which as

a whole will exhibit the functional property of that construct.

Dr. Robert Samuel Langer, who is an American engineer and the David H. Koch Institute Professor, is one of the pioneers

of tissue engineering technique. Organs which are successfully engineered can be mentioned to be artificial windpipe, liver,

bladder, kidney, lung, heart, bone, cartilage, penis, and skin. Tissues like vascular grafts, heart valves, and bone marrow

have also been generated using the technique. The basic strategy used by scientists to bioengineer tissues utilizes a

biomaterial as a structural and mechanical scaffold into which a specific cell population is incorporated. Growth factors and

other bioactive molecules can be added to the construct. After a period of maturation either in vivo or in a bioreactor, the

Bioengineered miniature human liver

( Source: http://www.gizmag.com/)

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anticipated end product is a tissue-engineered organ that serves as a functional replacement for damaged or missing tissue.

The first article published in The New York Times: A First: Organs Tailor-Made With Body’s Own Cells, assayed the

path-breaking research done in biomedical field which successfully developed hollow organs like bladders and windpipe.

The innovative research was credited to Dr. Paolo Macchiarini, who created a new windpipe by using plastic and the cells

of his patient, at the Karolinska Institute. But this was only the beginning of the technique and formation of complex organs

such as liver, kidney, pancreas, bone etc, was still a complicated procedure until the stem cell therapy came into existence.

On 3 July 2013, Nature, international weekly journal of science, published an article entitled Vascularized and functional

human liver from an iPSC-derived organ bud transplant, which covered the work of a Japanese research team which

for the first time derived liver from human pluripotent stem cell and cultured with developmentally important progenitor

cells self-organize into functional, three-dimensional liver buds. This proof-of-concept demonstration of organ-bud

transplantation provided a promising new approach to study regenerative medicine.

Tissue engineering of complex organs is now becoming a most promising long term treatment of tissue damage repair and

replacement therapy. Bioartificial pancreas is efficient in treatment of endocrine disorders like diabetes. To generate an

artificial pancreas in-vivo, a mesh of fibres along with encapsulated clusters of islet cells, semipermeable coating and

biocompatible outer layer, all are clubbed together to create the bioartificial pancreas device. The function of fibres is to

provide strength; the islet cells are encapsulated to avoid immune response; the semipermeable coating ensures the normal

diffusion of nutrients and hormones i.e. active biomolecules; and the biocompatible outer layer is to keep them all together

without inducing fibrotic response. This artificial pancreas can, therefore, contain islet cells which would behave like an

endocrine pancreas and secrete insulin, glucagon and amylin, thus treating the endocrine disorder.

Similar efforts have been made to create bioartificial heart by Taylor and colleagues, who generated a bioartificial heart

which had a heart structure with appropriate cell composition and cardiac pump function using this attractive technique in

2008. They perfused heart with 1% SDS, which is a strong ionic detergent, to produce acellular scaffolds, preserved

extracellular matrix and original microvascular network, and then repopulated these heart-like constructs with neonatal

cardiomyocytes and aortic endothelial cells. When provided with the suitable physiological stimuli, the recellularized organ

in the culture was able to further mature and display beating behaviors. Rhythmic contractions were observed by day 4,

pump function generated by day 8 and cardiac architecture was formed, as confirmed by histological analysis. The

bioartificial heart pumped with an amazing function, which was equivalent to about 2% heart power of an adult or 25%

heart function of a 16-week fetus. Uygun and colleagues modified Taylor’s perfusion decellularization technique to achieve

transplantable liver grafts in 2010.

Niklason and colleagues, in the same way, regenerated lung grafts using the decellularization and recellularization strategy

in 2010. In this regeneration technique, they removed cellular components from lung matrix, preserved the hierarchical

branching structures of the airways and vasculature bed in the extracellular scaffold, and reseeded pulmonary and vascular

endothelial cells in the acellular lung matrix.

In cases of burnt victims, artificial skin implantation is very common now-a-days. In recent studies, highly innovative

progress in the development and clinical implementation of highly sophisticated tissue engineered skin so as to increase

their medical safety, elasticity, durability, biocompatibility, and clinical efficacy has been reported. There are numerous

types of skin derived seed cells which can be used for the bioengineering of artificial skin. These cells include

Keratinocytes, Dermal fibroblasts, epidermal stem cells, melanocytes, non-cutaneous cells like embryonic stem cell,

mesenchymal stem cell, endothelial, amniotic and Inducible pluripotent stem cells (iPSCs). iPSCs for instance, as

mentioned earlier, were first discovered by Yamanaka and colleagues, who received The Nobel Prize in Physiology

Medicine, along with John B. Gurdon, in 2012 for the finding that mouse fibroblasts could acquire properties similar to

those of embryonic stem cells after "reprogramming”. Procedures differentiating mouse iPSCs into epidermal

keratinocytes are similar to keratinocyte differentiation of ESCs. By using the iPSCs-derived keratinocyte lineage cells,

Bilousova et al., have successfully regenerated epidermis, hair follicles, and sebaceous glands in the skin of athymic nude

mouse.

Nanotechnology is now-a-days, being coupled with tissue engineering to give rise to certain novel and innovative

opportunities in this research area. Nanomaterials have emerged as promising candidates in producing scaffolds able to

better mimic the nanostructure in natural extracellular matrix and to efficiently replace defective tissues. They also provide

a promising attachment, proliferation and differentiation of the stem cells in the device. Nanoscience, thus, is one of the

major future aspects of this technology where nanomaterials are used as biomaterial scaffolds to provide the framing

structure to the construct. Of course stem cell innovations have facilitated the technique in many ways and are another

rising aspect of artificial organ formation. It is expected that the combination of stem cells and nanomaterials will develop

into an important tool in tissue engineering for the innovative treatment of many diseases.

As we can see now, organogenesis is not a dream anymore and we are at the edge of improving human health by

eradicating and minimizing risks of fatality due to organ failure or impairments. With the emergence of tissue engineering

technique, science has once again proved that human brain is powerful enough to invade the most challenging health risks

including cardiac and endocrine disorders. Definitely, there are issues and risk factor in creating and transplanting an

artificial organ, but at this current stage of evolution, this technique for sure, has created a paving path leading to a

promising future.

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REFERENCES:

1. Fountain, Henry. A First: Organs Tailor-Made With Body’s Own Cells. New York Times. 15 Sept.

2012. http://www.nytimes.com/2012/09/16/health/research/scientists-make-progress-in-tailor-made-

organs.html?pagewanted=all&_r=0}

2. Kristine C Rustad, Michael Sorkin, Benjamin Levi, Michael T Longaker, and Geoffrey C Gurtner. Organogenesis.

2010 Jul-Sep; 6(3): 151–157. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946046/

3. Huanjing Bi , Yan Jin. REVIEW ARTICLE, Current progress of skin tissue engineering: Seed cells, bioscaffolds, and

construction strategies. Burns and Trauma. (1)(2). 2013. 63-72.

4. Anthony D Metcalfe and Mark W.J Ferguson. Tissue engineering of replacement skin: the crossroads of biomaterials,

wound healing, embryonic development, stem cells and regeneration. PubMed central. 4(14. 2007 June 22. 413–437.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373411/

5. Anne Trafton. Tissue engineering: Growing new organs, and more. MIT News. 14th

December 2012.

http://web.mit.edu/newsoffice/2012/engineering-health-tissue-engineering-growing-organs-1214.html

6. Tissue Engineering. Nature Biotechnology. (18). 2000.

http://www.nature.com/nbt/journal/v18/n10s/full/nbt1000_IT56.html

7. Takanori Takebe et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature.

(499). 25th

July 2013. 481-484. http://www.nature.com/nature/journal/v499/n7459/full/nature12271.html

8. Tissue Engineering -- Growing New Organs, and More: Research Could Lead to Better Ways to Heal Injuries and

Develop New Drugs. Science news. Science Daily. 14th

December 2012.

http://www.sciencedaily.com/releases/2012/12/121214095433.htm

9. Liu et al. Review, Generation of functional organs from stem cells. Cell Regeneration. (2)(1). January 2013.

http://www.cellregenerationjournal.com/content/2/1/1

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MOLECULAR DIAGNOSIS OF CANCER Vartika Rai

1 and Pranita Roy

2*

1M.Tech student,

2Assistant Professor

Amity Institute of Biotechnology,

Amity University Noida, India

proy@amity.edu

Abstract

Cancer that has a reputation of a deadly disease all around the world is a broad group of diseases that involves uncontrolled

cell growth and proliferation. The best method for its prevention and control is its early diagnosis and treatment. Before

molecular diagnostics, scientists relied on the results obtained from biopsies and microscopic examination of affected

tissue samples but nowadays molecular diagnosis is emerging as a new and eye opening approach that merges genomics

and proteomics for early detection and diagnosis of cancer. Genomics and proteomics tools are used to detect different

molecular signatures like genetic and epigenetic signatures, changes in gene expressions, protein biomarker profiles and

other metabolite profile changes, which in turn allows to identify the combinations of biomarkers which may detect best

the presence or risk of cancer or monitor cancer therapies.

Molecular diagnosis uncovers the different changes that occur during the transformation of a normal cell to a tumour cell

and capture this information as expression patterns. Robotically printed microarrays, Real time PCR and mi RNAs are

widely used techniques for measuring these expression pattern and help researchers to differentiate between a normal and a

cancer cell. Molecular diagnosis through proteomics make use of surface enhanced laser desorption/ionization time of

flight mass spectrometry and peptide receptors in mapping of protein patterns that are involved in malignant growth.

Nanotechnology is an evolving science that can be successfully used for cancer diagnosis in future. Nanoscale devices

quantum dots and carbon nano-tubes can be promising nano-tools for effective measurement of malignancy. All these

techniques offer great promise for revolutionizing the diagnosis of cancer. This article recaps some scientific

considerations about different relevant molecular diagnostics of cancer and consideration about future challenges.

Keywords: Molecular diagnostics, molecular signatures, cancer biomarker.

1. INTRODUCTION

“You can see a person's whole life in the cancer they get.” - Haruki Murakami

Cancer, a broad group of various diseases, is an outcome of alterations in critical regulatory genes that control cell

proliferation, differentiation and survival. The genetic changes lead to alteration in the signaling proteins along different

signaling pathways, disrupting the normal cellular pathways (1). As a result of the breakdown in the signaling pathway

cells divide and grow uncontrollably, forming malignant tumours, and invade nearby parts of the body. The cancer may

also spread to more distant parts of the body through the lymphatic system or bloodstream (A). To determine the causative

factors of cancer is a complex process. Many things are known to increase the risk of cancer. These include tobacco use,

certain infections, lack of physical activity, radiation obesity and environmental pollutants. There are around 200 different

known cancers that affect humans (1).

The development of cancer is a multistep process in which normal cells gradually progress to malignancy. The complete

sequence of events required for development of any human cancer is not yet known, but it is becoming interestingly clear

that both the activation of oncogenes (genes which promote cell growth and reproduction) and the inactivation of tumor

suppressor genes (genes which inhibit cell division and survival) are critical steps in tumor initiation and progression

(Figure 1). Genetic changes leading to cancer can occur at different levels and by different mechanisms (2). An error in

mitosis can lead to gain or loss of an entire chromosome. More common are mutations which are changes in the nucleotide

sequence of genomic DNA.The challenge for cancer diagnosis is to identify these genes and proteins at a very early stage.

This has lead to an exponential expansion of our understanding and knowledge, especially at the genetic, molecular and

cellular level (3, 4, and 5).

2. REVIEW OF LITERATURE Cancer is difficult to treat and within a decade despite of heavy funding to cancer research it has become a leading cause of

death in industrialised countries. It is a complex disease that varies widely in terms of its causes, development, and

progression (3, 5). At the molecular level, the problem of how to reduce cancer mortality is approached principally in the

following three ways:

a) Identification of patients who are liable to develop cancer genetically (detection of germ line mutations in patients)

and detection of genetic changes in somatic cells that favor degeneration to form cancer cells.

b) Early diagnosis of cancer is the best way for prevention and effective treatment as advanced cancer is difficult to treat

and has a poor prognosis.

c) Development of new molecular approaches that target the tumor cell or the tumor environment.

2.1 CANCER AND SIGNALLING PATHWAYS

Cancer is an outcome of an altered signaling protein along different signaling pathways. As a result of the breakdown in

the signaling pathway cells divide and grow uncontrollably, forming tumors. The most common signaling pathways that

have been shown to be implicated in cancer include the JAK/STAT pathway, the MAP-Kinase/ERK pathway and the NF-

kB pathway.

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2.1.1 THE JAK/STAT PATHWAY IN CANCER

The JAK/STAT pathway in response to growth promoting factors and cytokines plays an important role in mediating cell

fates, such as apoptosis, differentiation and proliferation. Upon binding their receptors, receptor-associated Janus Kinases

(JAKS) phosphorylate tyrosine residues of the receptors, as well as the Signal Transducers and Activators of Transcription

(STATs). The phosphorylated STAT bind to other phosphorylated STAT and thus forms homodimers. These translocate

to the nucleus and participate in transcriptional regulation of a variety of genes (6). Deregulation of JAK/STAT signaling

pathway can contribute directly and indirectly to tumorogenesis (7). It is reported that mutations, fusions, and/or

amplification of JAK/STAT signaling components such as the HER2/neu- in mammary and stomach carcinomas, or

Epidermal Growth Factor-Receptor (EGF-R) in breast, brain and stomach tumors, can confer hypersensitivity to mitogenic

signals and promote proliferation (B). Furthermore, STAT3 is constitutively activated in several major human carcinomas

and some hematologic tumors. Notably, STAT3 is persistently active in over 50% of lung and breast tumors and more than

95% of head and neck cancers. Impaired STAT signaling could therefore also indirectly contribute to tumor formation by

compromising tumor immune surveillance (8).

2.1.2 THE MAP KINASE/ERK PATHWAY IN CANCER

The RAS-Mitogen Activated Protein Kinase (MAPK) pathway promotes cell adhesion, proliferation, migration and

survival. This pathway plays an integral part in transducing signals from cytokines and growth factors through Receptor

Tyrosine Kinases (RTK). Central to this signaling cascade is RAS, a small membrane-bound GTPase switch proteins that

shuttles between two conformational states: active GTP-bound and inactive GDP-bound (8). It is activated by SOS which

is a guanine exchange factor usually found in the cytoplasm of the cell. Activated GTP-bound RAS carries the activation of

serine/threonine kinase RAF that activates mitogen-activated protein kinases (MAPK). It is also called extracellular signal-

regulated kinase (ERK). ERK1/2 translocates to the nucleus and activates the Jun/Fos transcription factors leading to cell

signalling (8).

Because of its central role in cell proliferation, deregulation of the SOS-Ras-Raf-MAPK signaling cascade leads to a broad

spectrum of human tumors. Most of these mutations occur in RAS and RAF and result in constitutive pathway activation

resulting in hyper proliferative state. RAS mutations are found in 45% of colon cancers and 90% of pancreatic cancers.

RAF mutations, in turn, are found in roughly two thirds of all melanoma. Consequently this pathway is a formidable target

for therapeutic intervention and has received tremendous attention (8).

2.1.3 NF-κB PATHWAY IN CANCER

The nuclear factor-kappa-B (NF-κB) pathway regulates genes involved in key cellular processes such as proliferation,

stress response, innate immunity and inflammation. In vertebrates, the NF-κB transcription factor family consists of

p50/p105, p52/p100, c-Rel, RelA and RelB that regulate transcriptional expression of hundreds of target genes. P105 and

p100 are proteolytically processed to give rise to p50 and p52, respectively. c-Rel, RelA, RelB, p50 and p52 can form

homo- and heterodimers, shuttle to the nucleus where they bind DNA regulatory κB sites (8). In the absence of signaling,

NF-κB dimers are located in the cytoplasm and inactivated by their interaction with I-κB inhibitory proteins. NF-κB

signaling is activated by a variety of extracellular factors such as the tumor necrosis factor-α (TNF-α), interleukin-1,

growth factors, bacterial or viral infections, oxidative stress and pharmaceutical compounds. In response to such stimuli, I-

κB is rapidly phosphorylated on serine 32 and 36 by the I-κB kinase (IKK). Phosphorylated I-κB is ubiquitinated by the E3

ubiquitin ligase complex and targeted for degradation by the 26S proteasome. The liberated NF-κB dimers can then

translocate to the nucleus and activate transcription of target genes (8).

Mutations and miss-regulation of NF-κB signaling has been involved in a variety of cancers, for example human B-cell

malignancies. The human REL gene, encoding one of the five NF-κB transcription factors is amplified in approximately

50% of Hodgkin’s lymphoma, approximately 10–20% of non-Hodgkin’s B-cell lymphomas and approximately 40%

natural killer T-cells lymphomas. It has been suggested that amplification of the REL gene and over expression of the

protein outcompetes the inhibitory effects of I-κB in the cytoplasm, leading to constitutive transcription of NF-κB target

genes and increased mature B-cell proliferation and survival. Consistent with this model, over expression of human REL is

sufficient to transform and immortalize primary chicken lymphoid cells in culture, whereas diminished levels of REL

inhibit B-cell proliferation. Immunohistochemical analysis of patient-derived lymphoma samples with REL amplifications

has confirmed nuclear REL expression in several cases (8).

2.2 MOLECULAR DIAGNOSIS OF CANCER

The most effective way to deal with cancer would be to prevent development of the disease. Many cancers can be cured by

localised treatments, such as surgery or radiation, if they are detected before they metastasize into the body. The initial

diagnosis of most cancers is through screening by the appearance of signs or symptoms which does not lead to a definitive

diagnosis, and thus requires the examination of a tissue sample by a pathologist. Before molecular diagnostics, clinicians

categorized cancer cells according to biopsy samples and by observing their appearance under a microscope (4). Molecular

analysis of the oncogenes and tumor suppressor genes involved in particular types of tumors has the potential of providing

information that is useful in the diagnosis of cancer and in monitoring the effects of treatment. Nowadays molecular

diagnosis is emerging as a new and eye opening approach that merges genomics and proteomics for early detection and

diagnosis of cancer.

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Merging two new disciplines, genomics and proteomics, molecular diagnostics categorizes cancer using technologies such

as microarrays and mass spectrometry. Genomics is the branch of science which deals with study of all the genes in a cell

or organism, while proteomics is the study of all the proteins. Through molecular diagnostics the interaction of genes and

proteins in a cell can be determined (Figure 2). Molecular diagnostics uncovers the sets of changes in a normal cell and

captures this information as expression patterns (9). The expression patterns are also called as "molecular signatures,"

which help in improving the clinicians' ability to diagnose cancer (A). Genomics and proteomics tools are used to detect

different molecular signatures like genetic and epigenetic signatures, changes in gene expressions, protein biomarker

profiles and other metabolite profile changes, which in turn allows to identify the combinations of biomarkers which may

detect best the presence or risk of cancer or monitor cancer therapies(10).

2.2.1 CANCER DIAGNOSIS AND GENOMICS Genomics applies recombinant DNA, DNA sequencing methods, and bioinformatics to sequence, assemble, and analyze

the function and structure of the complete set of DNA within a single cell of an organism (9, 10). The field is used to

determine the entire DNA sequence of organisms.

2.2.1.1 CANCER DIAGNOSIS THROUGH MICROARRAYS

A major emphasis in molecular diagnosis is on the use of DNA microarrays also called as “gene chips” for determining the

expression patterns of genes. The major challenge is to find the genes active in cancer and separate them from all of the

others in a cell. DNA microarrays can be used to compare the patterns of gene expression in two different cell populations,

such as a population of cancer cells with a population of normal cells (Figure 3). In this case, two different fluorescent dyes

are used (A). Microarrays allow researchers to "see" the expression of hundreds or thousands of genes all at once and their

comparison simultaneously. The results obtained from microarray experiments are dramatically changing cancer-treatment

decisions (9).

Cancer research make use of certain specific standard arrays which include lymphochip and other arrays that contain

strands of DNA called single nucleotide polymorphisms, or SNPs ("snips"), which are common variations in DNA. (Figure

4) DNA Microarray technology measures the relative activity of previously identified target genes. In 2003, Louis M.

Staudt studied gene expression profiling (the expression) of thousands of genes at once, to create a global picture of

cellular function and showed that the diagnosis of the hematologic cancers is commonly based on morphologic evaluation

supplemented by analysis of a few molecular markers. The process of expression profiling makes use of robotically printed

DNA microarrays to measure the expression of tens of thousands of genes at a time (Figure 5). Different analytic

techniques are used to classify cancers on the basis of their gene-expression profiles. These methods have revealed

unexpected subgroups within the diagnostic categories of the hematologic cancers that are based on morphology and have

demonstrated that the response to therapy is dictated by multiple independent biologic features of a tumor (11).

In recent years scientists have focussed on machine learning algorithms for classification and diagnosis of cancer based on

expression profiles. In 2003, Ringnér and Peterson surveyed the application of machine learning algorithms. They

emphasized on Microarray-Based Cancer Diagnosis with Artificial Neural Networks. Supervised methods that have been

applied to molecular classification of cancer tissues include correlation-based classification methods, artificial neural

networks (ANNs) and support vector machines (SVMs) (12).

However one of the difficulties in using gene expression profiles to predict cancer is how to effectively select a few

informative genes to construct accurate prediction models from thousands or ten thousands of genes. For solving this

problem in 2009 Wang and Gotoh conducted a research on Microarray-Based Cancer Prediction Using Soft Computing

Approach. They screened highly discriminative genes and gene pairs to create simple prediction models involved in single

genes or gene pairs on the basis of soft computing approach and rough set theory. Accurate cancerous prediction was

obtained when they applied the simple prediction models for four cancerous gene expression datasets: Central Nervous

System (CNS) tumor, colon tumor, lung cancer and diffuse large B-cell lymphoma (DLBCL). Some genes closely

correlated with the pathogenesis of specific or general cancers were identified (13).

Microarrays can also be used to detect differences in patterns of gene activity even within the same tumor type, as a single

type of cancer categorized microscopically can be of more subtypes, each with a distinct gene expression pattern. (Figure

6, 7)

2.2.1.2 CANCER DIAGNOSIS THROUGH MICRO RNAs

Micro RNA (abbr. miRNA), a small non-coding RNA molecule encoded by eukaryotic nuclear DNA, found in plants and

animals, functions in transcriptional and post-transcriptional regulation of gene expression (14). miRNAs function via

base-pairing with complementary sequences within mRNA molecules, usually resulting in gene silencing via translational

repression or target degradation (15, 16). The human genome may encode over 1000 miRNAs,(17) which may target about

60% of mammalian genes(18,19) and are abundant in many human cell types.

In 2006 Harris et al. conducted a research on the use of unique microRNA molecular profiles in lung cancer diagnosis and

prognosis. They examined MicroRNA (miRNA) expression profiles for lung cancers to investigate miRNAs involvement

in lung carcinogenesis. miRNA microarray analysis identified statistical unique profiles, which could discriminate lung

cancers from noncancerous lung tissues as well as molecular signatures that differ in tumor histology (20). In 2007,

William CS Cho presented a review on the importance of OncomiRs: the discovery and progress of microRNAs in cancers

stating the function of miRNAs as tumor suppressors and oncogenes, and designating it as oncogenic miRNAs

(oncomiRs). It has been reported that miRNAs play a crucial role in the initiation and progression of human cancer (21). In

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2008 Chen et al. reported on characterization of microRNAs in serum as a novel class of biomarkers for diagnosis of

cancer and other diseases and the dysregulated expression of microRNAs (miRNAs) in various tissues is associated with a

variety of diseases, including cancers. He and his colleagues demonstrated that the miRNAs are present in the serum and

plasma of humans and other animals such as mice, rats, bovine foetuses, calves, and horses. The levels of miRNAs in

serum are stable, reproducible, and consistent among individuals of the same species. They used Solexa to sequence all

serum miRNAs of healthy Chinese subjects and identified specific expression patterns of serum miRNAs for lung cancer,

colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases (22).

2.2.1.3 CANCER DIAGNOSIS THROUGH REAL TIME PCR

A quantitative polymerase chain reaction (qPCR), also called real-time polymerase chain reaction, is a laboratory

technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and

simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, quantitative PCR

enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount

when normalized to DNA input or additional normalizing genes.

In 2006 H.C.Dawn et al. conducted a research on multigene Real-time PCR Detection of Circulating Tumor Cells in

Peripheral Blood of Lung Cancer Patients. He and his team took a panel of 51 lung tumors and 13 normal lung tissues and

assayed them by the 4-gene multiplex RT-PCR to determine the specificity of the test for lung cancer. CLCA2, HMGB3,

L587S and ASH1 were identified in lung cancer tissues using genetic subtraction, microarray and quantitative PCR, and

found to be specific and complementary for detection of non-small cell lung carcinoma (NSCLC) and small cell lung

carcinoma (SCLC). These data indicated the diagnostic and prognostic utility of a multigene real-time RT-PCR assay to

detect tumor cells in the peripheral blood of lung cancer patients (23).

A recent study has been conducted on breast cancer diagnostics using RT PCR where the her-2(Human Epidermal Growth

Factor Receptor 2) gene quantification using immune histochemically-identified biopsies was done. A group of workers

reported that her-2 gene amplification and its over-expression in breast cancer cells is directly associated with aggressive

clinical behaviour (24). HER2 also known as Neu, ErbB-2, CD340 (cluster of differentiation 340) or p185 is a protein that

in humans is encoded by the ERBB2 gene. HER2 is a member of the epidermal growth factor receptor (EGFR/ErbB)

family. Amplification or over-expression of this gene has been shown to play an important role in the pathogenesis and

progression of certain aggressive types of breast cancer and in recent years it has evolved to become an important

biomarker and target of therapy for approx. 30% of breast cancer patients ( 8, B).

2.2.2 MOLECULAR DIAGNOSIS AND PROTEOMICS Beyond genes molecular diagnosis also deals with the investigation of proteome (all proteins produced by a cell). The

study of these proteins is proteomics. Cancer proteomics is enabling mapping of the patterns of proteins involved when

normal cellular pathways are hijacked in support of malignant growth. In cancerous tissue, some of the proteins critical for

normal communication are damaged, inactive, overactive, or missing entirely. The full set of deranged and dominating

proteins at work disrupting cellular communications may vary from one cancer type to another. They may also vary

somewhat from one patient to another within a cancer type (A). Cancer researchers are devising clever ways to capture

data on each cancer's characteristic unruly sets of proteins. For proteomic study the biopsy or blood samples from cancer

patients are used as the starting point to extract and analyze the various sets of interactive proteins (Figure 8). For many

proteins in the cell, phosphorylation acts as a switch that activates the protein. This active cell's phosphorylation state is

capture in proteomic study which gives an accurate pattern of proteins interacting in a cancer cell. The proteins are

prevented from getting deactivated by quickly treating the samples with enzymes to block the removal of phosphates from

proteins enabling researchers to identify a protein pattern almost identical to what was in the cell at the time of collection

(A).

2.2.2.1 CANCER DIAGNOSIS THROUGH ANTIBODY MICROARRAYS

One of the most widely used methods in cancer diagnosis by proteomics is through antibody microarrays. In 2003, Miller

et al. developed a practical strategy for serum protein profiling using antibody microarrays and applied the method to the

identification of potential biomarkers in prostate cancer serum. They compared protein abundances from 33 prostate cancer

and 20 control serum samples to abundances from a common reference pool using a two color fluorescence assay. Having

defined a set of reliable microarray measurements, they identified five proteins (von Willebrand Factor,

immunoglobulinM, Alpha1-antichymotrypsin, Villin and immunoglobulinG) that had significantly different levels between

the prostate cancer samples and the controls. These developments enabled the immediate use of high-density antibody and

protein microarrays in biomarker discovery studies (25).

2.2.2.2 CANCER DIAGNOSIS THROUGH SELDI-TOF-MS

Surface-enhanced laser desorption/ionization (SELDI) is an ionization method in mass spectrometry that is used for the

analysis of protein mixtures. SELDI is typically used with time-of-flight mass spectrometers and is used to detect proteins

in tissue samples, blood, urine, or other clinical samples. Comparison of protein levels between patients with and without a

disease can be used for biomarker discovery. In 2003 Veenstra et al conducted a study on cancer diagnosis using proteomic

patterns. In their research they described a revolutionary approach i.e. proteomic pattern analysis. It relies on the pattern of

proteins observed and does not rely on the identification of a traceable biomarker. Hundreds of clinical samples per day

can be analyzed utilizing this technology, which has the potential to be a novel, highly sensitive diagnostic tool for the

early detection of cancer. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF

MS) is the technology used to acquire the proteomic patterns to be used in the diagnostic setting (26 ). In 2005, Zhang et al

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used SELDI TOF MS for the detection of breast cancer. They developed and evaluated a proteomics approach to searching

for new biomarkers and building diagnostic models. SELDI-TOF-MS Protein Chip was used to detect the serum protein

patterns of 49 breast cancer patients, 51 patients with benign breast diseases, and 33 healthy women. Four candidate

biomarkers of breast cancer were found. The high sensitivity and specificity achieved by this method show great potential

for the early detection of breast cancer and facilitation of discovering new and improved biomarkers (27). Similarly in 2005

Yang et al conducted a research on application of serum SELDI proteomic patterns in diagnosis of lung cancer. The results

of their experiments suggested that serum SELDI protein profiling can distinguish lung cancer patients, especially Non-

small-cell lung carcinoma (NSCLC) patients, from normal subjects with relatively high sensitivity and specificity, and that

the SELDI-TOF-MS is a potential tool for the screening of lung cancer. (28).

2.3 CANCER DIAGNOSIS THROUGH PEPTIDE RECEPTORS Jean Claude Reubi in 2003 presented a review on peptide receptors as molecular targets for cancer diagnosis and therapy.

Peptide receptors can be used successfully for in vivo targeting of human cancers. In the review the author summarized and

critically evaluated the in vitro data on peptide and peptide receptor expression in human cancers. This data was considered

to be the molecular basis for peptide receptor targeting of tumors. The paradigmatic peptide somatostatin and its receptors

were extensively reviewed in the light of in vivo targeting of neuro-endocrine tumors. This information relates to

established and potential clinical applications in oncology (29).

2.4 CANCER DIAGNOSIS AND NANOTECHNOLOGY: FUTURE PERSPECTIVE

Cancer nanotechnology is an interdisciplinary area of research which covers a vast and diverse array of devices like

nanovectors for the targeted delivery of anticancer drugs and imaging contrast agents. Nanotechnology has evolved with

broad applications for molecular imaging, molecular diagnosis, and targeted therapy of cancer. It plays an important role in

realizing the goal of detecting transforming cell populations early by in vivo imaging or ex vivo analysis (B). This allows

the appropriate combination of agents to be chosen (based on accurate biological information on the tumour), targeting of

these agents (while avoiding biological barriers) to the early cancer lesions to eliminate or contain them without collateral

effects on healthy tissue, and monitoring the treatment effect in real time. The basic rationale involved in cancer-

nanotechnology is that nanometer-sized particles, such as semiconductor quantum dots and iron oxide nanocrystals, have

optical, magnetic, or structural properties that are not available from molecules or bulk solids (C). When linked with tumor

targeting ligands such as monoclonal antibodies, peptides, or small molecules, these nanoparticles can be used to target

tumor antigens (biomarkers) as well as tumor vasculatures with high affinity and specificity. In the size range of 5–100 nm

diameter, nanoparticles also have large surface areas and functional groups for conjugating to multiple diagnostic (e.g.,

optical, radioisotopic, or magnetic) and therapeutic (e.g., anticancer) agents. With minimal cancer cell sample preparation,

substrate binding to even a small number of antibodies produces a measurable change in the device’s conductivity, leading

to a 100-fold increase in sensitivity over current diagnostic techniques (30). Nanoscale cantilevers, microscopic, flexible

beams resembling a row of diving boards, are built using semiconductor lithographic techniques. These can be coated with

molecules capable of binding specific substrates-DNA complementary to a specific gene sequence, for example. Such

micron-sized devices, comprising many nanometre-sized cantilevers, can detect single molecules of DNA or protein.

Quantum dots, nanoscale crystals of a semiconductor material such as cadmium selenide, are another promising nanoscale

tool for laboratory diagnostics (30). Nanowires and nanocantilever arrays are among the leading approaches under

development for the early detection of precancerous and malignant lesions from biological fluids (Figure 9).

Recent advances have led to bioaffinity nanoparticle probes for molecular and cellular imaging, targeted nanoparticle

drugs for cancer therapy, and integrated nanodevices for early cancer detection and screening. These developments raise

exciting opportunities for personalized oncology in which genetic and protein biomarkers are used to diagnose and treat

cancer based on the molecular profiles of individual patients. Multifunctional nanodevices hold out the possibility of

radically changing the practice of oncology, perhaps providing the means to survey the body for the first signs of cancer

and deliver effective therapeutics during the earliest stages of the disease.

3. DISCUSSION AND CONCLUSION Molecular diagnostics is a rapidly-advancing area of research and medicine, with new technologies and applications being

continually added for rapid diagnosis of cancer. The technologies that come under the umbrella of molecular diagnosis

include gene expression profiling using microarrays, mi RNAs, SELDI TOF MS, proteomic analysis through peptide

receptors and antibodies. In the future, nanotechnology will have a broad impact in the field of molecular diagnosis of

cancer, which employs various nanodevices, DNA probes, second-generation biochips, micro fluidics etc. for early

detection and effective treatment of cancer. Molecular diagnostics for examining the expression pattern of cancer cells

offer great promise for revolutionizing our approaches to screening, diagnosis, and classification for many different types

of cancer. All these techniques have led to the discovery of various therapeutic molecules for cancer treatment, and the

optimization of drug therapy. No doubt molecular diagnosis will lead to significant improvements in care for cancer

patients in the not-too-distant future.

REFERENCES

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11. Staudt, L.M. (May 2003), Molecular Diagnosis of the Hematologic Cancers. N Engl J Med 348, 1777-85.

12. Ringnér M and Peterson C (March 2003), Microarray-Based Cancer Diagnosis with Artificial Neural Networks.

BioTechniques. 34, S30-S35.

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Informatics 2009, 7 123–139.

14. Chen, Kevin; Rajewsky, Nikolaus (2007).The evolution of gene regulation by transcription factors and microRNAs

Reviews Genetics 8 (2), 93–103.

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16. Kusenda B, Mraz M, Mayer J, Pospisilova S (November 2006). MicroRNA biogenesis, functionality and cancer

relevance. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 150 (2), 205–15.

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Spector Y, Bentwich Z (July 2005), Identification of hundreds of conserved and non conserved human microRNAs.

Nat. Genet. 37 (7), 766–70.

18. Lewis B.P., Burge C.B., Bartel D.P. (2005), Conserved seed pairing, often flanked by adenosines, indicates that

thousands of human genes are microRNA targets. Cell 120 (1), 15–20.

19. Friedman RC, Farh KK, Burge CB, Bartel DP (January 2009), Most mammalian mRNAs are conserved targets of

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Calin G.A., Chang-Gong Liu, Croce C.M., and Harris C.C.(MARCH 2006), Unique microRNA molecular profiles in

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Barbara K.Z. (2006), Multigene Real-time PCR Detection of Circulating Tumor Cells in Peripheral Blood of Lung

Cancer Patients. ANTICANCER RESEARCH 26, 1567-1576.

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quantification using immunohistochemically-identified biopsies. ONCOLOGY LETTERS 5, 295-298.

25. Miller J.C., Zhou H, Kewkel J, Cavallo R, Burke J, Butler E. B 3, The B.S., Haab B.B.(2003), Antibody microarray

profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers. Proteomics

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cancer. Current Opinion in Biotechnology, 15, 24–30.

27. Hu Y, Zhang S, Yu J, Liu J, Zheng S. (August 2005), SELDI-TOF-MS: the proteomics and bioinformatics

approaches in the diagnosis of breast cancer. The Breast Volume 14, Issue 4, Pages 250-255.

28. Shaun ying Yang, Xue-yuan Xiao, Wang-gang Zhang, Li-juan Zhang, Wei Zhang, Bin Zhou, Guoan Chen and Da-

cheng He (2005), Application of serum SELDI proteomic patterns in diagnosis of lung cancer. BMC Cancer, 5, 83.

29. REUBI J.C. Peptide Receptors as Molecular Targets for Cancer Diagnosis and Therapy. (2003), Endocrine Reviews

24(4), 389–427.

30. Ferrari M. (MARCH 2005) Cancer Nanotechnology: Opportunities and Challenges Nature Reviews. Volume 5. Pg no.

161 – 171.

ONLINE LINKS:

A. http://www.cancer.gov/cancertopics/understandingcancer/moleculardiagnostics.

B. http://en.wikipedia.org/wiki/HER2/neu

C. www.springer.com/Nanotechnology

Want to enjoy the status & royalty of published author? Write to us at publications.giap@gmail.com Get your theses / project / dissertation / case studies published in ISBN digital books. Get your research paper / opinion / article / blogs published online on www.giapjournals.com

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FIGURES

Figure1: Cancer requires multiple mutations (A).

Figure 2: Molecular diagnostics of cancer (A).

Figure 3: Gene expression analysis with the help of microarrays and differentiation between a cancer and normal cell (A).

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Figure 4: Different models of microarrays (A).

Figure 5: Cancer specific gene expression (A).

Figure 6: Cancer Subtyping(A) Figure 7: Use of microarrays in diagnosis of lymphomas (A).

Figure 8: Protein mapping (A) Figure 9: Quantum Dots

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RECTROACTIVE INHIBITION AND ITS AFFECT ON MEMORY Ritu Rani

1*, Ketki Sharma

2* and Aparna Sarkar

3

1, 2*

M.Sc Medical Physiology Students, Amity Institute of Physiology & Allied Sciences 3Associate Professor, Amity Institute of Physiology & Allied Sciences, Amity Institute of Physiotherapy

Amity University Uttarpradesh ,Sector -125, Noida, India.

medicalphysiologyamity@gmail.com, asarkar@amity.edu, dr_aparna_sarkar@yahoo.co.in

1. INTRODUCTION

Memory is an ability to store, retain and recall information and experience. In other words, it is also explained as the process of

encoding storage and retrieval of information. Encoding refers to transformation of information in form of codes. Storage is the

process of putting the coded information.

There are three types of memory basis of collecting and storage of information:

a. Sensory Memory- We receives several information from our environment through different sense organ at a particular time.

We pay attention to some information and reject others. The duration had here is for few seconds.

b. Short term memory- Once we pay attention to the selected information it is passed on to the short term memory. The

duration had here is for 30 seconds. The best example of this memory is the "serial position effect" if subject are said to listen

to certain words and then recall them instantly, it has been found that the subject will recall those items which appeared at the

end (which is known as regency effect)and the beginning (which is known as primary effect) of the list. The items encountered

most recently are remembered well.

c. Long term Memory- Some of the information reaching short term memory is processed by being rehearsed, is by having

attention focused on it perhaps by constantly repeating the words. The duration of information stored in long term memory can

be for life time. The information processed and stored here is systematically organized.

2. HYPOTHESIS

H0- Null Hypothesis:

There is no effect of nature of task given on the percentage of retroactive interference.

H1- Alternate Hypothesis:

If the interpolated task is similar to the original task, Retroactive inhibition will be more.

3. AIMS AND OBJECTIVE

a. To check whether task similarity and dissimilarity has any effects on retroactive inhibition

b. Measure the comparison between, Retroactive & Proactive Inhibition.

4. LITERATURE REVIEW

Retroactive inhibition is one aspect of the theory of interference. Interference Theory states that people forget not because

memories are actually lost from storage but because other information gets in the way of that people want to remember.

Retroactive inhibition occurs when material learn later destructs retrieval of information learnt earlier so old information

overlap with new information. For the study of the same an electronic memory drum is used for most of the 20th

centuries the

memory drum standard American apparatus for memory research. The first date from 1887 in the work of G.E. Muller and

Schwmann Cattell used something similar for his experiment.

The memory drum is a standard method of presenting paired associate and serial syllable list to participate in psychological

study of memory from 1890‟s until more modern computerized version came in. Muller and Schwmann,1894 discovered that

more time was necessary to relearn a series of non sense syllables if the stimulance syllables had been associated with other

syllables in the mean time for their result, the deduced the law of association and inhibition which is quoted by K line. “If „A‟

is already connected by „B‟, then it is difficult to connect it with „K‟ because „B‟ gets in the way”. Non sense syllables were

around by Shepherd and Fogelsonger in 1913 in a series of experiments in association and inhibition. In the experiment

changes were introduced to produce inhibition the findings were so great in many cases to present novel situation. This was

shown by introspection.

“Retroactive inhibition is a function of similarity between original task and interpolated task”.

Retroactive interference has been localized to the left anterior ventral prefrontal cortex. In adult‟s age 56-70 years showed less

magnetic activities in their prefrontal cortices than the control group.

Retroactive interference has been investigated using three ways:

i. Pitch Perception

Pitch perception used as a learning medium in investigation of Retroactive inhibition. It is found that the presentation of

subsequent stimuli in successation causes a decrease in recalled accuracy. Massaro found that the people forget because

presentation of successive auditory tones, confused perceptual short term memory, causing Retroactive interference as the

other information gets in the way and inhibits the retrieval of previously heard tones.

ii. Motor movement

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Wohldmann, Healey and Bourn state that Retroactive interference affects the retention of motor movements. The Retroactive

inhibition affects the performance of old motor movements when new motor movement gets.

iii. Word Task

Retroactive inhibition increase when items are similar. Control condition through the list of A against to error less conjugative

Hals and had little retroactive inhibition when asked to recall it after a period of unrelated activity.

5. HISTORICAL BACKGROUND

In 1885, Ebbinghaus found that the effect of memory can be seen more programmatically with the help of nonsense syllables

(i. e. meaningless words like MUS, DUV, TUM). He founds that if the subjects are given to memorize a list of given words and

then asked to recall them after certain period. The subject could recall nonsense syllables more effectively and quickly in

comparison to the meaningful words. He was latter on criticized by Bartlett (1932) who said that it is not only the nonsense

syllables, but cognition, social and experimental environmental which equally effect memory. So, these aspects should also be

taken care of while measuring memory. Several ways of measuring were developed later on today.

6. DIMENSIONS OF THE CASE STUDY

There is 5 lists in this case study i.e. 4 lists of nonsense syllables List A, List B, List D, List E & 1 list of meaningful words.

Each list contains 10 syllables.

LIST A LIST B LIST C LIST D LIST E

KZL CKP BOOK NRB TLM

BNQ KNZ GIRL XLQ VKR

XTP SKP SHOW WKP ZOP

DHR LNS TIME ZHM XPN

VKL BKF IDEA DYF FJG

FMC HDR BILL GRK WKR

JWS JXL HIGH CTJ NCR

FKN WQG NEAR SQV MDL

VDR RZL HOPE LVR ONQ

NOX TDX MEAN MZK SWF

There is no specific time limit for this but within recalling each 2 list there is 12 minutes break two lists.

7. PROCEDURE

Preparation

i. Materials required like memory drum checked whether in good condition, attach list in such a way that the subject could see

only one syllable at a time.

ii. Prepare all the 5 lists is for non sense syllable and 1 for meaning full words.

iii. Pencil eraser, plain papers, stopwatch include all above items kept readily.

Environmental Preparation

i. Make comfortable sittings arrangements.

ii. Adequate light and well ventilated room. The experiment is conducted in classrooms, the tables and the chairs arranged in

distance to the other group.

iii. Make sure that there is less noise so that the subject should not for difficulty while recalling.

Establishing Rapport

Rapport establishment is very important form of any test or experiment.

i. Informed the procedure to the subject after the informal taken subject's profile.

ii. Explain how to use memory drum.

iii. Explain time limits, intervals etc.

iv. The subject for control groups is very enthusiastic ask for the procedure.

v. My subject is Ms. Hemlata and she has told that she was very weak in recalling the things,

The experimental subject is very cooperative and silent person. Ms. Hemlata has also understood till the aspects of procedure.

Those of have assured their queries and make them ready to undergo the experiments.

Consent

i. Concentrate fully on recalling the list.

ii. If u exhausted in between recalling, stop for a while.

iii. You will get 12 min. break after recalling the list.

iv. Trials will be taken until you will fully recall/100% syllables for 2 lists.

v. Do not get distracted to the other things while recalling.

Precautions

i. Proper environmental preparation is very essential otherwise the subject could be distracted and it may hamper memory

function.

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ii. Prepare list properly/neatly, so that the every single syllable could be properly visible.

iii. Engage the subject during 12 min. breaker distract his attention other than the list items, so that the subject should not

continue to memorize lists.

iv. If the subject feel bored or want to quit then encourage him/her.

Introspective Report

After completion of the experiment the subject is interviewed. She feels that it was a difficult task for her. She was

comfortable but didn't want to take repeat trials.

According to my observation Ms. Hemlata is a hard worker and sincere in her work. She have tried to recall even she is not

very good in recalling things.

Ms. Hemlata is silent/shy person but very good in recalling things. She said that she enjoyed the experiment but sometimes

trials are bored. According to her body language she didn't get distracted, but cooperative and sincere in her efforts.

Interpretation

Case study for retroactive inhibition whether task similarity or dissimilarity has an effect on retroactive inhibition. Meaningful

words are very easy to recall then the non sense syllables and the same time when the similar task is given recalling retroactive

inhibition will be more.

8. CONCLUSION

We have conducted a case study on memory dated 22/6/2013 at Jamia Milia Islamia University using memory drum. It is a

apparatus which cylindrical in shape and can be rotated clockwise for the serial presentation of the syllable or words for

regulated period.

One person has given similar task to recall and another person is giver is given dissimilar task and result are proven the H1

hypothesis to be true i.e., one similar task is given there is 50% retroactive inhibition.

REFERENCES

1. Neel, Ann (1977). Theories of Psychology: a handbook, Cambridge.

2. Wohldmann, E.L., Healy, A.F., Bourne Jr., L.E. (2008). A mental practice superiority effect: Less retroactive interference

and more transfer than physical practice. Journal of Experimental Psychology: Learning, Memory, and Cognition.

3. Greenberg, R.; Underwood, B.J. (1950). "Retention as a function of stage of practice". Journal of experimental

psychology.

4. Jonides, J.; Nee, D.E. (2006). "Brain mechanisms of proactive interference in working memory". Neuroscience.

5. Sternberg, RJ. (2009). Applied Cognitive Psychology: Perceiving, learning and Remembering. London.

6. http://en.wikipedia.org/wiki/Interference_theory

7. Galotti, K.M. (2008), Cognitive Psychology, Perception, Attention and Memory, London.

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BioEvolution

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ROLE OF PCR IN DIAGNOSTICS Amit Kumar

1, Ritu Rani

2 and Arti Goel

3*

1M.Sc Clinical Microbiology Student

2M.Sc Medical Physiology Student, Amity Institute of Physiology & Allied Sciences

3Assistant Professor, Amity Institute of Microbial Biotechnology,

Amity University, Noida, India

amitkumartech89@gmail.com, chauhanritu4u@gmail.com,

*agoel2@amity.edu, peena_agrawal@yahoo.co.in

1. INTRODUCTION PCR or Polymerase chain reaction is a laboratory procedure in which million of copies of a specific piece of DNA are made.

PCR is an amplification method, where by the tiniest amounts of DNA that may be present in blood, hair or tissues can be

copied so that there is enough for analysis.

The name of this method is derived from the key component involved that carries out the replication of the DNA, called a

DNA Polymerase. This is an enzyme that exists in nature.

2. ROLE OF PCR IN DETECTING INFECTIOUS AGENTS PCR is used in analyzing clinical specimens for the presence of infectious agents, including HIV Hepatitis Human,

Pailomavirus, Epstein-Barr Virus, malaria and anthrax. PCR in particularly invaluable in the early deletion of HIV as it can

identify the DNA of the virus within human cells immediately following infection, as opposed to the antibodies that are

produced weeks or months after infection. PCR can also be used to determine the viral load.

Malaria is traditionally diagnosed by identifying malaria parasite (Plasmodium falcipurum) through microscopic analysis of the

blood but PCR technique has been useful in that it can vapidly identify the malarial parasite species.

PCR can be used for the identification of causative agent of Anthrax that is Basillus authracis. PCR has become an important

tool in determination of the need to rapidly diagnose. PCR replaces the culture methods which take to much time. This is a

more rapid, sensitive and sensitive method. PCR also permits to identify non-cultivated or slow-growing microbes like

anaerobic bacteria, mycobacteria or viruses from tissue culture. The basic for PCR diagnostic applications in microbiology is

the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

3. IMPORTANCE OF PCR IN CANCER DIAGNOSTIC

Number of cancers is characterized by small mutations in certain genes. PCR is an invaluable as it can provide information on

a patient’s prognosis and predict response. It can also be applied in detecting leukaemia patients following treatment by

counting the number of cancerous cells.

4. IMPORTANCE OF PCR IN GENETIC DISEASES AND PATERNITY TESTING

The important application of PCR is to analysis of mutations that occur in many genetic diseases like sickle cell anaemia,

muscular dystrophy, cystic fibrosis etc. PCR can be done from a single cell taken from an embryo before birth because of very

sensitive.

The PCR is also can applied for paternity testing. A swap from inside the cheek is taken of both child and parents. The DNA is

extracted from the cell obtained and is analysed by PCR. The DNA of everybody is the same in all the cells of body. A child’s

DNA should have part of the DNA mother’s and father’s both. Several locations called “LOCI” on the sequences of these

“LOCI” are compared to the mother and watch from both parents.

5. IMPORTANCE OF PCR IN HISTOPATHOLOGY

For the molecular genetic research the histopathology lab is a vital source. The observation skills of the histopathologist can be

combined with molecular genetic data that are molecular genetic data that are readily gained by the use of the PCR.

In the diagnostic histopathology, important areas are those in which diagnosis is difficult by the use of conventional

morphological and immune-phonotypic methods and in which molecular aberrations that can readily be detected are strongly

associated with diagnostic categories of disease. These are currently met in two areas.

a. Lymphomas

b. Sarcomas

a. Lymphomas

PCR is widely used in the field of study and development of diagnostic technique in lymphomas and leukaemias.

Firstly, it has enabled the cumbersome gene rearrangement techniques of southern blot analysis to be replaced by rapid in

expensive strategies for clonality analysis and the detection of chromosome aberrations that can be applied to paraffin wax-

embedded specimens.

Secondly, it has simplified amplification and sequence analysis of immunoglobulin and T-cell receptor genes and his has

greatly improved our understanding of the biology of the lymphomas and provided clone specific markers for the study of

dissemination, progression and response to therapy.

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b. Sarcomas

These are a group of aggressive tumors in which the diagnosis on histological grounds alone may be very problematic many

sarcomas carry recurrent chromosome translocations that can be used as diagnostic markers. The RT-PCR is the most practical

and vital used method and analysis of this translocation has now become a routine part of the diagnosis of the sarcomas in a

number of labs.

6. IMPORTANCE OF PCR IN FORENSIC SCIENCES

Modern forensic science uses PCR analysis of highly polymorphic regions of the genome, which permits identification of

individual. DNA profiling is a technique used for identify someone passed on their DNA profile. PCR is an important method

in genetic finger printing which can indentify and one person from millions of others. The real time fluorescence based

quantities PCR (qPCR) has become the benchmark technique for the detection of nucleic acids in every field of microbiology,

biomedical research, biotechnology and in forensic application.

Finally, a common pattern in different individuals other than identical twinge is extremely unlikely.

7. FUTURE PROSPECTIVE OF PCR

Now-a-day PCR has become well established method for the study of nucleic acids sequences in histological specimen or

material. The challenge is to put the technology to full use by identification of the genetic changes that are important in clinical

disease. Knowledge of these changes will permit the establishment of rapid, cost-effective test for diagnosis and monitoring of

disease as well as providing a basic understanding of their underlying causes. These developments will be enhanced by our

unraveling of the complete human genome and by improvements in techniques for gene amplification using histological

material.

The standardization of techniques by number of centers collaboration are required the ensure that the PCR and related

techniques are used to their full potential in patient health management.

REFERENCES

1. Principal of gene manipulating by B. Primrose, Richard M. Twyman, R. W. Old.

2. Medical Lab Technology by Prafull B. Godkar.

3. Medical biology and Biotechnology by John M. Walker, Ralph Rapley.

4. An introduction to molecular biotechnology, molecular fundamentals, methods, application in molecular biotechnology by

Michael Wink.

5. Molecular Diagnostic by Georg P. Patrinos, Wilhelm Ansorge.

6. Microbiology and Pasrasitology by V. N. Panda

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