Cryopreservation

Cryopreservation is a technique of preserving and storing viable biological samples in a frozen state over extended periods of time. The preservation of animal cell has become dependent upon using low temperature to render the cell metabolically inert. The term ‘cryopreservation’ refers to ‘a method of cold storage.’ The optimum temperature for preserving animal germplasm is liquid nitrogen temperature of -196 degrees Celsius. Temperatures from -70 to -80 degree Celsius may be sufficient enough to maintain cell viability for a few months. Cryobiology is the study of the effects of extremely low temperatures on biological systems such as cells or organisms.

Definition

Cryopreservation is the process of cooling and storing cells, tissues and organs at very low temperatures to maintain their viability. Spermatozoa were the first mammalian cells to be cryopreserved successfully (Polge et al 1949).

Cryopreservation methods

The two most commonly used cryopreservation methods for animal germplasm are slow programmable freezing and vitrification.

Slow programmable freezing – In slow freezing, cells in a medium are slowly cooled to below freezing point. Slow cooling is needed in order to increase osmotic strength, which causes an efflux of water from the cells. Machines are used to freeze biological samples  using programmable sequences i.e. ‘freezing down’ a sample to better preserve it for eventual thawing before it is frozen or cryopreserved in liquid nitrogen. Biological samples such as oocytes, skin, blood products, embryo and stem cells are preserved by slow programmable freezing machines.

Vitrification – The term ‘vitrification’ refers to any process resulting in ‘glass formation’(‘arrested liquid state’) the transformation from a liquid to a solid in the absence of crystallization (solid-liquid).  Vitrification usually requires the addition of cryoprotectants prior to cooling. The cryoprotectants act like antifreeze: they decrease the freezing temperature. They also increase the viscosity. Instead of crystallizing, the syrupy solution becomes an amorphous ice- it vitrifies.

Stroage temperature

Cells can be stored for varying lengths of time at temperatures between -70 to -196 degrees Celsius. For short –term storage (few weeks) cells can be preserved at -70 degrees Celsius in standard mechanical freezers. For long-term storage, cells can be preserved at liquid nitrogen containers.

Liquid nitrogen containers

Liquid nitrogen containers are basically robust, heavily insulated vessels into which liquid nitrogen is poured at regular intervals in order to maintain the required temperature. Cells can be stored in either vapour phase – containers at -120 degrees Celsius or liquid phase containers at -196 degrees Celsius of liquid nitrogen. Storage at -120 degrees Celsius does not significantly reduce cell viability. But indefinite storage of cells requires liquid phase containers.

Cryoprotective agents (CPAs)

Glycerol and DMSO are the most commonly used cryoprotective agents. Fetal bovine serum (FBS) is employed in mammalian cryopreservation solutions, but it is not a cryoprotective agent. Dextrans, sorbitol, trehalose, polyethylene glycols, starches, sugars, and polyvinylpyrrolidone provide considerable cryoprotection in a variety of biologic systems (Mazur 1981).  Salts, such as magnesium chloride, have been reported to be cryoprotective agents (Karow and Carrier 1969). Cryoprotectants protect slowly frozen cells by one or more of the following mechanisms: suppressing high salt concentrations; reducing cell shrinkage at a given temperature;reducing the fraction of the solution frozen at a given temperature and minimizing intracellular ice formation. Combinations of cryoprotectants may result in additive or synergistic enhancement of cell survival (Brockbank and Smith 1993, Brockbank 1992). Intracellular cryoprotectants of low molecular weights permeate cells e.g. glycerol, dimethyl sulfoxide. Extracellular cryoprotectants with relatively high molecular weights do not penetrate cells e.g. polyvinylpyrrolidone, hydroxyethyl starch.

Advantages of cryopreservation

Germplasm cryopreservation of the sperm, eggs and embryos contributes directly to animal breeding programmes. Germplasm cryopreservation also assist the ex situ conservation for preserving the genomes of threatened and endangered species. Cryopreserved sperm, oocytes and embryos are used for artificial insemination and embryo transfer in the livestock industry. Cryopreservation also has enormous applications in the artificial propagation of widely diverse aquatic organisms. Cryopreservation of  sperm and embryonic cells has been successful in a number of teleosts and invertebrate species.  The establishment of germplasm banks using cryopreservation can contribute to conservation and extant populations in the future. Cryopreservation provides a continuous source of tissues and genetically stable living cells for a variety of purposes including research and biomedical processes. Cryopreservation reduces the risk of microbial contamination or cross contamination with other cell lines. Cryopreservation reduces the risk of morphological or genetic changes. It also reduces costs of maintenance of cells, tissues or organs.

Safety considerations

The main dangers come from explosions of ampoules or cryotubes. The technician must wear appropriate protective clothing, protective goggles, face mask, insulated gloves etc. Careless handling of cold containers can also cause burns, so it is important to use handling tongues. Nitrogen gas is colorless, odourless and tasteless. It cannot be detected by the human senses. So liquid nitrogen can be used only in well ventilated areas.

Stages in the cryopreservation

Selection of cell lines -à cultivation of cells------à Screening of cells -----à preparation of cells-----à freezing of cells ---à Evaluation of viability.

Preparing cells for cryopreservation

Cells are prepared by trypsinization (0.25% w/v trypsin) to detach adherent cell from flask surfaces. Cells in suspension are centrifuged at around 100xg for 5to 10 minutes and the pellet is suspended in a small volume (1-2 ml) of storage medium.

Storage medium

The cryopreservation media generally consists of a base medium, protein source and a cryoprotective agent. The cryoprotective agent protects the cells from mechanical and physical stress and reduces water content within the cells. Cryoprotective agent minimizes the formation of cell-lysing ice crystals. The formation of ice crystals may disrupt the cell membrane leading to the death of the cells. The composition of the medium used to suspend cells contains a cryoprotectant (usually glycerol or dimethyl sulphoxide, DMSO) and a high protein concentration (serum 20% v/v) cryprotectant = 7-10 % (v/v); serum – 20% (v/v).

Freezing

Since freezing is stressful, the rate of cooling from 0 to 50 degrees Celsius must be slow and controlled. A fall of about 1 degree Celsius per minute is optimal. Rapid chilling results in thermal shock and leads to cell death or injury.  Once a temperature of -50 degrees Celsius is reached, the cells must be cooled rapidly to the final holding temperature.

Thawing of frozen cells

Cells retrieved from storage must be thawed rapidly to ensure maximum survival. The ampoule should be plunged into a beaker of water at 37 degrees Celsius. The cell suspension can be transferred drop wise into a container holding about 20 ml of pre-warmed growth medium supplemented with 10% fetal calf serum.

Conclusion

Cryopreservation of gametes, embryos and embryonic cells has become of immense value in animal biotechnologies which provide an important tool for protecting the endangered species and genetic diversity. Cryopreservation protocols have been introduced as techniques for germplasm preservation of vegetatively propagated horticultural and staple food crops. The establishment of cryobanks to utilize the cryopreservation worldwide would be a significant and promising task in the future.

Ultracentrifugation

Centrifugation is the most widely used technique for understanding cellular and subcellular structures. During centrifugation, solid particles experience a centrifugal force, which pulls those outwards i.e. away from the centre. The velocity with which a given solid particle moves through a liquid medium is related to angular velocity. In other words, centrifugation a procedure involves the use of centrifugal force for the sedimentation of components of a mixture. More dense components of the mixture move away from the axis of the centrifuge, while less dense components of the mixture move towards the axis. The sedimentation process is accelerated by the centrifugal field.

Definition of centrifuge

Centrifuge is an apparatus that rotates at high speed and by centrifugal force separates substances of different densities. In other words a centrifuge is a device for separating particles or macromolecules (e.g. cells, sub-cellular components, proteins, nucleic acids) from a solution according to their size, shape, density, viscosity of the medium and rotor speed.

Definition of ultracentrifuge

Ultracentrifuge is a high-speed centrifuge for separating microscopic and sub-microscopic materials to determine the sizes and molecular weights of colloidal and other small particles.

Principle of centrifugation

The principle of centrifugation is that an object moving in a circular motion at an angular velocity w is subjected to an outward force F through a radius of rotation r in cms expressed as F=w²r . F is frequently expressed in terms of gravitational force of the earth (RCF). The operating speed of the centrifuge is expressed as revolutions per minute ‘rpm’.

The velocity of the moving particle is expressed in the form of sedimentation coefficient (S) v = S (w²r). Sedimentation coefficient is characteristic constant of a molecule and is a function of size, shape and density. The rate of sedimentation can be increased by raising the revolutions per second.

General types of centrifuges

Low – speed centrifuge –(desk top or clinical centrifuges)- maximum speed 3000rpm. It is used for separating serum form blood or separation of RBC etc.

High speed centrifuge – 25,000 – 30,000rpm. It functions at low temperature of 0-4 degree Celsius. It is used for cell fractionation i.e. separation of organelles.

Ultracentrifuge – High speed at 75,000 rpm and refrigerated.  It is used for the separation of cell organelles.

Analytical centrifuge - it has a built in optical system to measure the sedimentation characteristics of macromolecules.

Components of a centrifuge

The principal components of a centrifuge are a rotor to hold sample tubes and an electric motor to spin the sample. There are 4 types of rotors: Swinging bucket rotor (horizontal orientation), fixed angle (30 degree to the axis of rotation), vertical tube (centrifuge tube orients at an angle of 90 degree).

Types of ultracentrifuges

There are two types: analytical and preparative models.

    Analytical ultracentrifuge – It consists of rotors and tubes called cells. The instrument is designed to allow the person to follow the progress of the substances in the cells, while centrifugation is in progress. By estimating sedimentation velocity during centrifugation, the molecular weight can be determined.

Preparative ultracentrifuge – It is used for the purification of the components of macromolecules and all determinations are made at the end of centrifugation. The instrument does not have a monitoring device. Centrifugation is accomplished by rotors which are either swinging bucket type or fixed angle rotors. Swinging bucket rotors in which the buckets become horizontal, while in motion. In fixed angle rotors, the tubes of the centrifuge are set at fixed angles and the rotor moves in a specified plane at all times.

Description of ultracentrifuge

The ultracentrifuge consists of a fixed angle rotor of aluminium or titanium revolving at high speed about an axis in an evacuated chamber. The solution containing macromolecules is taken in the cell having quartz windows.  The cell is almost filled with the solution and sealed to withstand the pressure developed in the intense centrifugal field. A beam of light is allowed to pass through the solution, which then falls on a detector – normally a photographic plate.  Since light passing through an area of the sample is proportional to the molecules present in that region, the darkening produced in the photographic plate indicates the concentrations at various depths of the centrifuge tube.

The ultracentrifuge can be useful in two different methods: sedimentation equilibrium and sedimentation velocity methods.

In sedimentation equilibrium method, equilibrium is attained between the rate of settling down of the molecules and at the rate at which they diffuse back because of the thermal motion and Brownian movement under action of gravity. This  method takes several days to complete because of the low centrifugal forces (10,000-100,000g).

In sedimentation velocity method, higher centrifugal forces (up to 500,000 times the gravity)  are applied to accelerate sedimentation. This method starts with a well defined boundary or layer of solution near the center of the rotation and follows the movement of this layer toward the outside of the cell as a function of time. When a solution containing uniformly distributed solutes is centrifuged at high speed (55,000rpm), the particles migrate outwards from the centre of rotation, forming a well defined boundary between the solvent portions with or without particles.

Analytical Ultracentrifugation (AUC)

All analytical techniques require the use of an ultracentrifuge and can be classified into differential centrifugation and density gradient centrifugation. The density gradient centrifugation is further subdivided as zonal and isopycnic centrifugation.

Differential centrifugation is a technique commonly used by biochemists. A tissue sample such as liver is homogenized at 32 degrees in a sucrose solution that contains a buffer. The homogenate is then placed in a centrifuge and spun at constant centrifugal force and at constant temperature. After sometime a sediment forms at the bottom of the centrifuge tube called pellet and the overlying solution is called supernatant. The overlying solution is then placed in another tube, which is then spun at higher speeds. 

Applications – Differential centrifugation is used to determine the number of components and number of species; detection of impurities, molar mass of species; kinds and stoichiometry of chemical reactions present in solution including association with ligands, self- association etc. Materials analyzed include macromolecules such as proteins, polysaccharides, nucleic acids; small molecules such as drugs, ligands, gases and large aggregates such as viruses and organelles.

Density gradient centrifugation

The separation is  done in a medium having different density gradients. The selection of gradient medium is an important prerequisite. The gradient medium should not affect the cell sample. The medium should be easily sterilizable, recoverable and non-corrosive. Most common media includes sucrose, glycerol, sorbitol etc. There are discontinuous and continuous density gradients. In discontinuous density gradient medium, the density increases one layer to another. This medium is useful in the separation of whole cells, sub-cellular organelles or lipoproteins. In continuous density gradient medium, the density decreases from the bottom of the solution to the meniscus. This medium is useful in the separation of ribosomes, viruses, proteins and enzymes.  Density gradient centrifugation can be of two types such as rate zonal centrifugation and isopycnic centrifugation.

Rate zonal centrifugation – centrifugation is carried out at a very low speed for a short time so that the particles settle down. However centrifugation should be stopped before the particles of any zone settle at the bottom e.g. separation of nucleic acids, ribosomal subunits.

Isopycnic centrifugation – Isopycnic means “of the same density.” Isopycnic = equal density and separation is on the basis of different densities of the particles. Molecules are separated on equilibrium position, not by rates of sedimentation. The particles of solution move according to their buoyant densities and become static at a place, where the density is greater than their own. This requires a very long time centrifugation and high speed. As an example, the pellet obtained by centrifugation of the tissue homogenate at 10,000g is suspended in increasing densities of sucrose solution and centrifuged for several hours at 40,000rpm. Now the individual organelles move to the region of their own equilibrium density and remain at the specific regions. This method is useful in the separation of proteins, intracellular organelles and nucleic acid fraction.

Applications of density gradients

Density gradients are widely used to separate and purify, on a preparative scale, a variety of cells, organelles and macromolecules such as nucleic acids or proteins. Gradients are required for analytical experiments for example to measure the apparent buoyant densities or sedimentation coefficients of particles; to estimate the size, conformation or turnover rates of proteins and nucleic acids; and to investigate the  effects of chemical, physical or biochemical treatment of the sample material.

Scientific research skills

Research can be defined as a careful and systematic investigation in some field of knowledge, undertaken to establish facts or principles. Scientific research is a continued search for scientific knowledge and understanding by scientific methods. Research is aimed at obtaining the information to test specific hypotheses.

6- basic steps in scientific method 

 1)Defining the problem by reviewing the relevant knowledge. Based on these activities, hypotheses are stated, questions are formed and experiments are designed. 2)Planning the research to collect the data required to evaluate the hypotheses or answering the research questions. 3)Carrying out the experiment to obtain the desired data. 4)Analyzing the results so that conclusions can be drawn.5) Interpreting the results so that practical applications can be made and 6)Reporting the results in a way that all relevant audiences will benefit from the knowledge obtained.

Methods of scientific research

There are three methods of scientific research such as descriptive method, experimental method and statistical method.

1. Descriptive methods 

They provide a description of the thing being studied. This method is very crude and it may be some combination of words and numbers. It can be a natural observation, systematic observation and by developing tests. Greeks first employed natural observation to record what they see. This method is unsystematic and time consuming. The research using systematic observation can be carried out by checklist technique, questionnaire method and public opinion.

2. Experimental methods

In this method, the experimenter changes or varies something by  keeping other conditions  as constant as possible and  looks for some effect of the changes or variations on the thing being studied.
Experimental design – it must be planned with great care to control various factors/ variables.

Variables – a variable is something that varies. It can be quantitatively measured. Variables fall into two classes: independent and dependent variables. An experiment must have at least one independent and one dependent variable. An experiment may also have more than one independent and more than one dependent variable. In a graph, the horizontal axis (abscissa) depicts the independent variable and the vertical axis (ordinate), the dependent variable.

1.       An independent variable is a variable that the experimenter selects and manipulates. In other words the variable that is purposely changed is an independent variable. Each change of a variable is known as a level of independent variable. Each Experimenter also selects the dependent variables.

2.       The dependent variable is a variable that changes as a result of changing the independent variable.

Constants – the various factors in an experiment that do not change.

Controls – the main point of doing an experiment is to compare control factors with experimental factors otherwise it is difficult to tell what is going on.

The scale – it is a measuring device which consists of a sequence of interchangeable units beginning with zero.

Ranking – it is the arrangement of the units of a measurable quality in the order of amount.

Rating – it is the arrangement of items into ascending or ordered classes.

3. Statistical methods

Statistical methods focus on the significance of differences, sampling error and probability. Correlation refers to a co-relationship between two sets of scores.  Reliability refers essentially to repeatability. Validity refers to what the test is supposed to be measuring.

Scientific reasoning

It refers to a body of techniques for investigating a phenomena and acquiring knowledge. It consists of systematic observation, measurement and experiment. Scientific methods requires intelligence, imagination and creativity. This method is an ongoing cycle of formulating, testing and modifying hypotheses.

Types of research design

Exploratory research (huh?) is conducted to generate or gather basic knowledge. It is performed to clarify relevant issues, uncover variables or simply to collect more information.

Descriptive research (who, what, where, how) is conducted to provide further insight into the research problem by describing the variables of interest.

Causal research (if...then) is done to provide information on cause and effect relationships.

Importance of scientific research

•  Develop new methods to conduct scientific research.
• Increase the sum total of information/ philosophies in various fields of science.
• Develop and apply new devices to conduct research.
• Increase the general availability of new materials and certain services.

Parts of a research article

Abstract –a brief overview of the article.
Introduction – the purpose of the present study.
Review of literature – present relevant literature related to the present scientific study and justifying the choice of the present study which is uncovered till date.
Methodology - define new terms and describe the instruments and procedures.
Result – report the findings with tables and figures.
Conclusions – support the present findings with relevant literature.
References - cited literature may reflect the investigator’s knowledge on the subject and validity of the present study with reference to relevant published information.
"Man is the interpreter of nature, and science is the right interpretation."

Human somatic gene therapy - concepts and applications

Human gene therapy can be defined as the transfer of exogenous genes or nucleotide sequences into somatic cells for the purpose of preventing, correcting or healing various diseases.

Somatic gene therapy is a new type of weapon in the fight against acquired diseases.DNA is considered as a drug providing a framework for curing thousands of genetic diseases. It also satisfies one of the greatest dreams of clinical medicine: ‘molecular surgery’ at the root of the disease.

Cystic fibrosis (CF)

People suffering from cystic fibrosis lack CF gene needed to produce a salt-regulating channel protein, cystic fibrosis transmembrane conductance regulator (CFTR). This protein regulates the flow of chloride in to epithelial cells that cover the air passages of the nose and lungs. Without this regulation, patients with CF disease build up thick mucus that makes them prone to chronic lung disease. The gene therapy technique to correct this abnormality might employ an adenovirus to transfer a normal copy of the CFTR gene. The gene is introduced into the patient by spraying into the nose.

Familial hypercholesterolemia (FH)

The patients with genetic disorder unable to process cholesterol properly by a low density lipoprotein receptor(LDLR), which leads to high levels of fat in the blood stream.  Patients with FH disease suffer from heart attacks and strokes because of blocked arteries. A gene therapy approach is developed with partial removal of patient’s liver. Corrected copy of a gene is inserted into liver sections, which are transplanted back into patients. The healthy gene may reduce the cholesterol build-up in the blood of the patient.

Duchene muscular dystrophy (DMD)

DMD is the most common childhood form of muscular dystrophy because of the failure to express dystrophin in the muscle fibres. It is a lethal, recessive X-linked disorder, more frequent in males. Muscular dystrophy is characterized by progressive muscle weakness, defects in muscle proteins and death of muscle cells and tissue. Viral mediated in vivo gene transfer method was developed to deliver dystrophin gene to patient’s muscle. Simple non-viral gene transfer in vivo was also developed using naked plasmid DNA.

Cancer therapy

In general, cancers have at least one mutation to a proto-oncogene (yielding) an oncogene) and at least one to a tumour suppressor gene allowing the cancer to proliferate. Oncogene inactivation may be targeted at the level of DNA, RNA transcription or protein product. Oligonucleotides are designed in sequence specific manner to target the promoter regions of oncogenes. At the RNA level, antisense techniques prevent transport and translation of the oncogene mRNA by providing a complementary RNA molecule (e.g., C-myc gene). Ribozymes, antisense oligonucleotides with a cleavage action will reduce the stability of oncogene mRNA. Restoration of the tumour suppressor gene such as p53 can be sufficient to cause cellular apoptosis and arrest tumour growth.

Molecular chemotherapy for cancer cells

Herpes simplex virus thymidine kinase (HSV-Tk) converts the prodrug ganciclovir into toxic metabolites. This toxicity is sufficient enough to kill the cancer cells.

DNA repair by gene therapy

Another approach involves the repairing of a gene using chimeric oligonucleotides. Homologous recombination is a natural process that controls the replacement of a defective gene. Highly precise DNA repair is performed by using DNA oligonucleotides to introduce site specific changes in the genome, even a single incorrect base can be corrected.

Transplantation tolerance in organ transplanted patients

In organ transplanted patients, the use of immunosuppressive drug is associated with increased risk of the development of cancer, infectious and ischemic heart disease. Gene therapy can be used to reduce the immunogenicity by introduction of genes to block T-cell activation.

HIV treatment by gene therapy

By introducing an antiviral gene into an infected T-lymphocyte, the HIV virus can be killed or expressing an antiviral gene in the normal T-lymphocyte may protect the patient from future HIV infection.

Replacement of hormones and blood factors by gene therapy

If an erythropoietin gene or factor IX gene is transferred via adeno-associated vector into muscles, it will cause the expression of relevant protein.

Prodrug activation

In herpes simplex virus thymidine kinase method, the thymidine kinase enzyme in every transduced cell converts the prodrug ganciclovir into monophosphate and triphosphates forms. The triphosphate form of ganciclovir interferes with the DNA replication and kill  the cancer cells.

Treatment of hepatitis B and C

The genetic sequence of RNA molecules of the particular virus is cut and destroyed by using ribozyme producing genes.

Environmentally sustainable development

Our society depends on the maintenance and protection of the environment. Ecological communities provide exergy (high quality energy), materials and information required for the human societies to sustain themselves. Urban development, agriculture, mineral/oil extraction, fisheries, and forestry practices can threaten the very existence of ecosystems and alter/eliminate important habitats, key species and people’s way of life.

Sustainability is the capacity to endure. Sustainability is a state of balance between resource use and the regenerative capacity of the earth. Sustainability lies in the interplay of environmental quality, economic vitality and social equity.

Environmental sustainability

Environmental sustainability refers to the maintenance of natural capital (e.g., natural resources). The term ‘natural capitalism’ was coined by Paul Hawken in 1994. Natural capital is equivalent to ecological wealth which refers to the resources and services provided by nature.  In other words natural capital is comprised of environmental resources and ecological services that can be used for life and factors of production. The human economy depends on the planet’s natural capital and the utilization of natural capital beyond its regenerative capacity results in depletion of the capital stock. The factors depleting natural capital include over-population, poverty, unsustainable resource use, environmentally degrading economic policies, and technological inputs.

The natural capital performs 3 distinct types of environmental functions:

1. Provision of resources for production - the raw materials that become food, fuels, metals, minerals, timber etc.
2.  Absorption of waste from production – both from the production process and from the disposal of consumer goods.
3. Basic ecological services – e.g., climate control, shielding of UV radiation by ozone layer, air/ water purification, water storage, nutrient/mineral cycling, soil renewal, waste treatment etc.

Concept of critical natural capital (CNC) – the concept originates from the idea that there is a certain minimal amount of natural capital necessary for ecosystems (ecosystem limits) to continue to function and provide services for its inhabitants. It is an ecosystem’s ability to support an adequate standard of living for human beings which includes drinking water, food, shelter, a moderate climate and resources for production.

It indicates human demand on the biological capacity of the earth.

Ecological sustainability

Ecological sustainability can be described as ‘securing quality of life within the limits of nature. Ecological sustainability is a conservation concept-meeting human need without compromising the health of ecosystems.

 It is the capacity of natural ecosystems to maintain their essential functions and processes and retain their biodiversity in full measure over a long period of time. Achieving ecological sustainability is a balancing act between current needs and future needs.

Sustainable development

Equity, security and the environment are the key  elements of the definition of sustainable development.Sustainable development can best be visualized in ‘the critical triangle of development’ with 3Es: environmental (ecological development), equity (social development) and economic development. Economic development has to do with the creation of material wealth (goods and services) to meet the human basic needs. Ecological development means protection and conservation of our natural resources. Sustainable should also guarantee inter and intra generation equality with respect to meeting all basic needs. In general sustainable economic development improves the economy without undermining the society and the environment.
  In 1983, the United Nations called for a high level commission, the World Commission on Environment and Development (WCED), commonly known as the Brundtland Commission. In 1987, its final report ‘our common future’ stressed the need for economic growth and development strategies in all countries that recognized the limits of the ecosystem’s ability to regenerate itself and absorb waste products.

Definitions of sustainable development

·       The sustainable development is defined as ‘forms of progress that meets the needs of the present without compromising the ability of future generations to meet their needs (our common future, 1987: The world commission on environment and development).

·       The sustainable development is defined as the maintenance of essential ecological processes and life support systems, the preservation of genetic diversity and the sustainable utilization of species and ecosystems (IUCN/WWF/UNESCO,1991).

·       The sustainable development is the improvement in the quality of human life within the carrying capacity of supporting ecosystems.

Concepts related to  sustainable development 

1.    Introduces the idea of a strong link between economic growth and natural resources/environment.

2.    Introduces the idea of a complex relationship between growth and the environment, drawing attention to the need of environmental sustainability, economic sustainability, social sustainability and the need for conciliation in conflicts between these different dimensions.

3.    Asserts that ‘zero’ economic growth can be as harmful to the environment as uncontrolled economic growth.

4.    Introduces the idea that the fight against poverty, for social justice and quality of life are essential aims in order to ensure sustainability in environmental, economic and social terms and

5.    Asserts the idea that sustainability is not a linear process and cannot be gauged against a single and developmental model.

Green economy

Green economy is an economic development model based on sustainable development and knowledge of ecological economics. This concept is often associated with ideas such as “low-carbon growth or green growth.” In green economy, the environment is an “enabler” of economic growth and human well being. Green economy includes green energy generation based on renewable energy as an alternative to fossil fuels and energy conservation for efficient energy use. Karl Burkart defines a green economy as based on 6 main aspects: Renewable energy, Green buildings, Sustainable transport, water management, Waste management and Land management. The Rio Declaration recognizes the “integral and independent nature of the Earth, our Home” and in principles 1 and 3 that humans are “entitled to a healthy and productive life in harmony with nature” and the development must “equitably meet developmental and environmental needs of present and future generations.”

Environmental distress syndrome

When the natural environment is subject to multiple stresses, it can exhibit distress symptoms.  The term ‘environmental distress syndrome’ refers to deteriorating environmental conditions and concomitant threats to human health.  In other words environmental distress syndrome is a condition that affected the human beings of the earth after years of pollution and exploitation of the planet. A distress syndrome refers to the irreversible processes of system breakdown leading to the termination of the system before its normal lifespan. An ecological system should be healthy and free from ‘distress syndrome’. Healthy ecological systems are an essential condition of healthy people, healthy communities and sustainable livelihoods.

Pollution, the introduction of contaminants into an environment that causes instability, disorder, harm or discomfort to the physical ecosystem or living organisms.

Paul Epstein (1997) of Harvard University’s centre for health and global environment lists 5 symptoms of environmental distress syndrome.

1.    The re-emergence of infectious diseases e.g., cholera, typhoid, dengue fever, drug-resistant tuberculosis.

2.    Loss of biodiversity e.g., decline of frogs in 140 countries from 6 continents.

3.    The growing dominance of generalist species –e.g., crows, Canada geese.

4.    The decline in pollinators e.g., bees, birds, bats, butterflies, beetles.

5.    The proliferation of harmful algal blooms e.g., paralytic shellfish poisoning.

Stress from human activity is a major factor in transforming healthy ecological systems to sick systems. The complex interaction of population, technology and human behaviour has resulted in anthropogenic stress on most of the world’s ecological systems (population-pollution syndrome).

Environmental stress

Environmental stress can be either natural or anthropogenic (i.e., resulting from human actions). Many natural environmental stresses such as hurricanes, droughts, floods, earthquakes and forest fires are a periodic feature of earth. But anthropogenic environmental stress includes the production and release of chemical compounds and large scale land-use changes result directly from human actions. The population explosion, agricultural expansion and industrial revolution greatly enhanced the anthropogenic stress on the environment. The intensities of ecological stresses vary in space and time. When the ecosystem is subjected to a chronic stress exceeding its tolerance limit, the ecosystem may display a syndrome of disruptions of its structure and function. The structural changes include biotic impoverishment with a reduction in size, number and abundance of organisms. The functional changes include gross community metabolism, efficiency of mineral cycles and changes in the energy flow rates.The stress in aquatic ecosystems is best exemplified by eutrophication (forced nutrient  enrichment), Increased primary production with algal blooms and insufficient decomposition of organic matter with increased anaerobic zones. There is a replacement of longer lived larger species by short – lived opportunistic species.

Progression of global environmental stress

All environmental changes progress at two levels:

Systemic global changes refer to changes operating at the global scale. For example, the doubling of carbon dioxide from more fossil fuels leads to enhanced greenhouse effect which leads to global climatic changes.

Cumulative global changes refer to the snowballing effect of local changes which add up to produce change on a global scale. E.g., acid rain or soil erosion.

An ecological system is healthy and free from ‘distress syndrome’ if it is stable and sustainable that is if it is active and maintains its organization and autonomy over time and is resilient to stress (Costanza, 1992).

Costanza’s concept (1992) of ecosystem health indicators

Costanza proposed 6 attributes of ecosystem health indicators.

1.    Homeostasis (self – regulation) 2. Absence of disease 3. Diversity or complexity (number and types of species) 4.stability or resilience 5. Vigour or scope for system growth and 6. Balance between system components.

Xu and Mage (2001) proposed 4 sets of criteria to assess ecosystem health: structural changes, functional changes, organizational changes and dynamics.

"The earth is what we all have in common."- Wendell Berry.
"In nature nothing exists alone." -Rachel Carson, Silent spring.

Environmental Education - objectives and importance

Environmental problems have become the issues of global concern due to their worldwide impact. A better understanding of one’s own environment is indispensable for its rational development. Environmental education (EE) is a powerful means to know and understand the physical and cultural environment as a whole with the rational use and conservation of environmental resources for development.

Definition

The international union for the conservation of nature (IUCN) has stated that ‘environmental education is the process of recognizing values and clarifying concepts in order to develop skills and attitudes necessary to understand and appreciate the interrelatedness among men, his culture and his biophysical surroundings’.

Environmental education is the cornerstone of long-term environmental strategies for preventing environmental problems, solving those which arise or have occurred and assuring environmentally sound sustainable development.

Aims of environmental education (UNESCO, Tbilisi declaration, 1978)

1.    To foster awareness and concern about environmental issues that affect us at local, regional, national and global levels.

2.    To provide every person with opportunities to acquire the knowledge, values, attitudes, commitment and skills needed to protect and improve the environment.

3.    To develop and reinforce new patterns of environmentally sensitive behaviour among individuals, groups and society as a whole for a sustainable environment.

5 – Objectives of Environmental education (UNESCO – UNEP Jan.1996)

1.    Awareness – to acquire an awareness and sensitivity towards the environment as a whole and the issues, questions, and problems related to environment and development.

2.    Knowledge – to acquire a basic understanding of the environment and its associated problems.

3.    Attitudes – to acquire social values, strong feelings of concern for the environment and the motivation to actively participate in protection of the environment.

4.    Skills – to acquire the skills for identifying and solving environmental problems.

5.    Participation – to develop a sense of responsibility and motivation to be actively involved at all levels in creating a sustainable environment.

Guiding principles of environmental education

(UNESCO-UNEP-IEEP-the Belgrade Charter, 1975)

·       Environmental education should be a continuous life-long process both in-school and out-of-school, both formal and non-formal.

·       Environmental education should be interdisciplinary in its approach.

·       Environmental education should emphasize active participation in preventing and solving pollution and environmental problems.

·       Environmental education should examine major environmental issues from a holistic point of view.

Global initiates on Environmental education

The UNESCO and UNEP created 3 major declarations that have guided the course of environmental education (EE).
Stockholm Declaration (Sweden, June 5-16, 1972)
It declared that EE must be used as a tool to address global environmental problems. The document published has 7 proclamations and 26 guiding principles regarding the preservation and enhancement of human environment.
The Belgrade Charter (Belgrade, October 13-22, 1975)
The charter added goals, objectives and guiding principles for new environmental programmes. It also added the general public in the programmes.
The Tbilisi declaration (Georgia/USSR, October 14-26 1977)
This declaration updated the goals, objectives, characteristics and guiding principles of environmental education.

Environmental teaching

Awareness and education are important inputs for a correct appraisal of environmental problems. Environmental education can be taught at various levels: formal and non-formal systems including adult education. In the formal level, there are 4 distinctive but chronological steps, which are mutually supportive.

Primary level-àlower secondary level--à higher secondary level--à tertiary level

At the primary level emphasis has to be on environmental awareness. At the lower secondary level, learners have to be taught to appreciate the real-life environmental situations at the local level. At the higher secondary level, Environmental conservation has to be emphasized. At tertiary level, learners have to be taught on sustainable development i.e., sustainable agriculture and forestry, integrated land use management, eco-farming and waste management etc.

Approaches of EE in formal education

1.    Infusion approach – the concepts and values of EE are infused with other subjects that are already included in the curriculum such as physics, chemistry, geography and languages. This approach is adopted at the school level in India.

2.    EE as separate subjects – Indian universities have introduced separate subject at undergraduate and post graduate levels.

3.    Occasional programmes for EE- Under this approach, occasional camps or eco-excursions are organized solely for the purpose of environmental education.

Conclusion

Education has always played a crucial role in the society because it inculcated necessary skills and attitudes in the learner’s life. So education has been identified as a critical driving force in environmental education. The content and approaches of environmental education need sufficient review and change at the various levels of formal, non-formal and informal education at all levels of society.

Nucleic acid probes - characteristics, probe-assays and applications

Nucleic acid probes are used to detect specific sequence of target DNA or RNA molecules from a mixture.  The single stranded soluble nucleic acid probe hybridizes to the complementary target DNA or RNA that is immobilized on a nitrocellulose or nylon membrane. The nucleic acid hybridization is used for a variety of biotechnological applications such as the detection of cloned DNA, analysis of genetic organization and the diagnosis of genetic diseases. Nucleic acid hybridization is rapid, specific and reliable between complementary strands.

         Definition

In DNA technology, a labelled single stranded nucleic acid molecule is used to tag a specific complementary nucleotide sequence in a nucleic acid sample.

         Kinds of nucleic acid probes

1.    Genomic DNA probes – using probes, more copies of fragments of genomes are obtained, purified and labelled.

2.    cDNA probes – obtained from mRNA using RTase enzyme and labelled.

3.    Synthetic oligonucleotide probes – made up of 14-20 base pairs long.

4.    RNA probes –to locate RNA molecules and was synthesized in vitro using phage RNA polymerase.

Characteristics of DNA probes

1.    DNA probes are very specific in their hybridization wit particular complementary DNA sequence.

2.    DNA probes never produce false positive or false negative results.

3.    DNA probes are more stable and resistant to heat and chemicals. They can be used to characterize DNA from dead tissues.

4.     The size of the DNA probes range from 13 – 100bp.

5.    With the help of PCR, DNA sequences can be amplified up to million folds.

              Labels of DNA probes

·       Labelled with radionuclides-P³², I¹²⁵, S³⁵, H³

·       With fluorescent labels – fluorescein, rhodamines, ethidium, rare earth chelates.

·       With luminescent labels – luminal derivatives, acridium esters, luciferase.

·       With enzyme markers – alkaline phosphatase, horse radish peroxidise.

·       With the combination of above labels

Preparation of DNA probes

The DNA sequence of interest is first cloned into a suitable plasmid. Then the plasmid with the cloned gene is transformed into appropriate host cells to amplify the plasmid. The transformant host cells are then selected using biomarker like antibiotic resistance of plasmids. The transformant host cells are separately cultured to get more copies of plasmid – DNA construct. Then the host bacterial cells are harvested and lysed in order to isolate the plasmid DNA with the cloned gene. The plasmid DNA lysate is precipitated differentially and centrifuged in Cesium chloride density gradients using ethidium bromide to purify plasmid DNA-DNA construct. Plasmid is purified and used for preparing DNA probe by incorporating appropriate label. The cloned gene can be retrieved by REase. The enzyme DNAase is used to cause nicks and DNA polymerase I is used to incorporate radio-labelled (P32 – dCTP) deoxyribonucleotides.  The DNA is denatured using sodium chloride to free out unincorporated deoxyribonucleotides from DNA’ by column chromatography using sephadex 600. Two peaks one with labelled DNA and other for P32 –dCTP are obtained. The first peak is separately collected.

DNA probe assays

The assay consists of 4 steps such as sample preparation, hybridization, separation and detection.

Sample preparation - the samples like blood, CSF, urine, stool or tissues are collected from pathogenic organisms. The separation of the target DNA molecules requires elaborate, time consuming multistep mechanical and chemical extraction procedures. The target molecules may be increased by PCR or LCR etc.

Hybridization – Specific DNA probes are added to the DNA derived from the sample and allowing complementary DNA to join. It may be carried out either as heterogeneous or homogeneous process.

Heterogeneous process uses solid support to immobilize target DNA. Examples are dot blot or in situ hybridization. Homogeneous process occurs in solution and after hybridization event, hybrid DNA is usually immobilized onto a support like filter or beads. Homogeneous process allows rapid and more efficient hybridization.

Separation – column chromatography, electrophoresis or paramagnetic beads may be used to recover true hybrids.

Detection techniques – radioisotopic, flurorescent or chemical luminescent techniques may be used. The probes are appropriately labelled with radioisotopes (I125, P32, S35 or H3) or flurorescent dyes or enzymes.

Applications  of DNA probes

Genetic engineering research - DNA probes are used to identify the clones possessing specific DNA sequences and to identify transformants possessing specific genetic characters. The probes can be used in basic studies in molecular biology, population genetics, and ecological genetics.

Disease diagnosis/epidemiological studies – DNA probes are used to identify genetic or microbial diseases or changes in the DNA of brain tumours.

Identification of food contaminants – it is used to identify pathogenic viruses in imported food samples.

Forensic investigations – Probes are used to identify criminals, solving disputed paternity cases, establishing family relations and solving immigration cases.

Agricultural applications – Probes are used to identify good varieties of seeds or plants.

Taxonomy - Probes are used to identify organisms at species or strain level.

DNA probes are used in anthropological survey of human races, and ante-natal diagnosis of inherited diseases.

Systems Biology - concepts, properties and insights

 Systems biology sees an organism as a living system and not as a machine. Living organisms, societies and ecosystems are all systems. Living systems form multi-leveled structures. Each level consists of several subsystems. The molecules in the subsystems combine to form organelles, which in turn combine to form cells. The cells form tissues and organs, which themselves form larger systems like digestive or nervous systems. They altogether present a ‘stratified order’ of organization from molecules to human beings. Then people form families, tribes, societies and nations.

What are natural systems?

All the natural systems are integrated wholes, whose specific structures arise from the interactions and interdependence of their parts. Most natural systems are hierarchical: they are composed of smaller sets of systems made up of smaller interacting parts. The properties of a system cannot be reduced to those of its parts. A living system is capable of adaptation and evolution. In the evolutionary process, there is a progressive increase of complexity, coordination and interdependence. The living systems exhibit self-maintenance which includes the processes of self-renewal, healing, homeostasis and adaptation. They also show self-transformation and self – transcendence, a phenomenon that expresses itself in the processes of learning, development and evolution.

The living organisms have an inherent potential for reaching out beyond themselves to create new structures and new patterns of behavior. The stability of a living system is continually tested by its fluctuations. When a system is disturbed, it has the tendency to maintain its stability by means of negative feedback mechanism. The stability of a living system is never absolute.

Example of a natural system

Planet earth is an integrated system consisting of 4 main subsystems. The lithosphere consists of rocks and minerals that form the solid body of the earth. The atmosphere has a layer of air surrounding the earth’s surface. The hydrosphere forms water on and near the earth’s surface. The biosphere is a layer of living organisms of which human beings form a part.

What is a system?

The word ‘system’ is defined as a group of interrelated, interacting or interdependent constituents forming a complex whole. The components work together to perform a function e.g., a computer.

There are three propositions in the definition: 1.the system is made up of component parts; 2.the parts work together; 3. The whole thing serves some purpose.

A system is a set elements that are orderly and interrelated to make a functional whole e.g., social system

Boundaries are the borders that separates one entity from another e.g., skin. Homeostasis is the tendency of a system to maintain a relatively stable, constant state of balance.

Systems view – looks at the world in terms of relationships and integration.

Systems thinking –it is process thinking.

Systems approach – emphasizes basic principles of organization.

Concept of holon and holarchy

Holon implies wholeness. A holon is something that is simultaneously a ‘part’ and a ‘whole’. The term holon was coined by Arthur Koestler from the Greek ‘holos’ meaning whole (all) and the suffix ‘-on’ meaning an individual part. This concept describes something that is whole in itself and, simultaneously a part of a larger system. So the main characteristic of a holon is the duality of being both an autonomous whole, while also being a part of a larger whole. This larger whole does only exist by the combination and the interactions of the composing parts holons. In other words a holon is a whole to those parts beneath it in a hierarchy but a part to those wholes above it. For example we can see an organization as a holon, because it is made up of smaller systems (e.g., people), but it is also a part of a larger system ( e.g., the community or economy).
A holarchy is conceived of as a hierarchical structure of holons. Our body, society, nature, the earth and the universe are all examples of so-called holarchies. The holarchy is the grouping of parts to create larger wholes where wholes are greater than the sum of the composing pats and each part in itself is also a whole.
                              Organisms-->organs-->tissues-->cells--->molecules.

Objective of systems biology

The world of nature is often very complex. To understand this complexity, scientists try to envisage ‘systems model’. Models in science tend to be simplified representations of reality that can be explained mathematically and through the use of graphics.

                                 Inputs----àSystem----àoutputs

Common characteristics of systems

1.    All systems have some structure and organization.

2.    They are all some extent generalizations, abstractions or idealizations of the real world.

3.    They all function in some way.

4.    There are, therefore functional as well as structural relationships between the units.

5.    Function implies the flow and transfer of some material.

6.    Function requires the presence of some driving force or source of energy.

7.    All systems show some degree of integration.

Properties of biological systems

1.    A biological/ natural system is intrinsically dynamic in nature. They show a high degree of internal flexibility and plasticity. It is this flexibility that enables living organisms to adapt to new circumstances.

2.    The functioning of organisms is guided by cyclical patterns of information flow known as feedback loops. When a system breaks down the break down is usually caused by multiple factors that may amplify each other through interdependent feedback loops.

3.    A living organism is a self-organizing system, which means that its order in structure and function is not imposed by the environment but is established by the system itself.

4.    Living organisms are open systems which mean that they have to maintain a continuous exchange of energy and matter with their environment to stay alive.

5.    Living organisms have a high degree of stability with dynamic properties. The stability consists in maintaining the same overall structure in spite of ongoing changes and replacements of its components.

6.    The phenomenon of self-organization is dynamic with self-renewal and self-transcendence. Self-renewal is the ability of living systems continuously to renew and recycle their components, while maintaining the integrity of their overall structure. Self-transcendence is the ability to reach out creatively beyond physical and mental boundaries in the process of learning, development and evolution.

7.    Fluctuations play a central role in the dynamics of self-maintenance. All variables of the system oscillate between a wide range of upper and lower limits so that the system is in a state of continual fluctuations, even when there is no disturbance. Such a state is known as homeostasis.  It is a state of dynamic and transactional balance. Negative feedback is only one aspect of self-organization through fluctuations.

Types of systems

Systems can be divided into open and closed systems depending on the inputs and outputs of energy.Traditionally systems can be 4 types like morphological, cascading, and input-output and control systems.

Black box system-we only understand inputs and outputs

Grey box system – we understand internal working

White box system – we understand individual components, their flows and storages.
Thermodynamic systems classification
An open system exchanges matter and energy with its environment.
A closed system exchanges only energy with its environment.
An isolated system exchanges neither matter nor energy with its environment.

Key traits of environmental systems

1.    Openness – it exchanges matter and or energy with other systems.

2.    Integration – refers to the strength of the interactions among the parts of the system and

3.    Complexity- how many kinds of parts a system has.

   Traits of an ecosystem

   An ecosystem is basically an energy processing and nutrient-regenerating system. Plants and animal populations within the system represent the subsystems. Inputs into the systems are both biotic and abiotic. The abiotic inputs are energy and inorganic matter. Radiant energy influences temperature, moisture, seasonality and photosynthesis. Inorganic matter consists of all nutrients like water, carbon dioxide, oxygen and so forth that affect the growth, reproduction and replacement of biotic material and maintenance of energy flow. The biotic inputs include other organisms that move into the ecosystem.