LYMPHOCYTE CULTURE

BY: Ezhuthachan Mithu Mohanan (MSIWM043)

Cells circulating in body and part of immune system are known as lymphocytes. There are mainly two main types of lymphocytes, which includes B Cells and T cells. 

Peripheral Blood Lymphocyte culture: 

PBLC is a technique used in Cytogenetics used for karyotyping analysis. The most convenient method to study chromosomes and by using cell culture technique it can be helpful for clinical and research purposes.  The powerful mitogen for human T cell is PHA (Phytohaemagglutinin).PHA is a plant hormone, which is obtained from Red Kidney bean. The function of PHA is to bind to T Cell membrane, which stimulates metabolic activity.  It is important to obtain cells at metaphase plate, since the study of Chromosome is possible and also easy to stain the chromosome at metaphase stage. During metaphase chromosomes are attached to spindle fibres.

Procedure for PBLC

  • Cell culturing should be done in a laminar Hood to avoid contamination. 
  • 1 ml of Venous blood should be collected 
  • Take 7 ml of RPMI1640 ( reconstituted with 10% fetal calf serum) in sterile culture tube
  • Add 0.5 ml blood in culture tube
  • Add 0.1 ml of PHA ( Previously thawed) 
  • Incubate this tube in sterile CO2 Incubator at 37°C FOR 69 hours
  • Add 40 µL of Colchicine at 69:00 hours
  • Incubate for 30minutes at 37°C
  • After Incubation centrifuge at 2000 rpm for 15 minutes
  • Remove supernatant and add 5 ml of Hypotonic solution
  • Mix gently and Incubate in waterbath for 37°C for 20 minutes
  • Add ice cold fixative (1:3 acetomethanol) and flush thoroughly
  • Centrifuge at 2000rpm for 15 minutes and remove supernatant
  • Repeat this above two steps for 3-4 times
  • Mix final suspension of 1 ml of fixative and make drop preparation in cold wet slide
  • Fix on flame and stain with 4% Giemsa
  • Observe and Scan for well spread metaphase plate in microscope

Function of each reagent

  1. PHA: it binds to membrane of T Cell and stimulate metabolic activity and cell division
  2. Colchicine: Suppress mitosis and cell division, but there is no interruption of chromosome multiplication
  3. Hypotonic solution : Used for swelling of cells
  4. Aceto-methanol: Helps in hindering cells at metaphase stage 

SPEMANN-MANGOLD ORGANIZER

                 By: N. Shreya Mohan (MSIWM042)

The Spemann-Mangold organizer are a consortium of cells that are required for the commencement of the neural tissue during the development of an amphibian embryo. Hilde Mangold, the then doctorate student along her mentor Hans Spemann, first published this work in 1924. This discovery gave so much scope to the developmental biology field, that this became one of the few doctoral theses to have won the Nobel prize in 1935. They showed that, of all the tissues in the early gastrula stage, only one has its fate determined. The overview of this organizer proved that destiny of the cells can be altered and influenced by factors from other cell clusters.

This self-differentiating tissue is the dorsal lip (the dorsal bordering region of the blastopore, which acts as the centre of differentiation) of the blastopore, the tissue derived from the grey crescent cytoplasm. When this dorsal lip tissue was transplanted into the so-called belly skin region of another gastrula, it not only continued to be blastopore tip, but also started the process of gastrulation and embryogenesis in the nearby tissues. Later on, two conjoined twins were formed instead of one. 

In this experiment, Spemann and Mangold used two different pigmented embryos from two newt species- that is, the darkly pigmented Triturus taeniatus and the non-pigmented Triturus cristatus. Two different species were taken for this experiment for transplantation by Spemann and Mangold as it would be easier to identify which one was the host and donor tissues respectively. At first, the dorsal lip of an early T. taeniatus gastrula was removed and then implanted into the area of an early T. cristalus gastrula was supposed to turn into ventral epidermis. As predicted, the dorsal lip tissue invaginated (cleaved) showing the qualities of self-determination and disappeared under the vegetal cells. The egg is divided into two regions- the animal pole (top part of the egg) and the vegetal pole (bottom part of the cell). Usually, the genetic material and proteins are unevenly distributed among these two poles.

The donor tissue (the pigmented species) of newt then continued to self-differentiate and divide into a structure called chordamesoderm (notochord) and other mesodermal structures respectively which usually comes from the original dorsal lip. 

Now, these newly made donor- derived mesodermal cells move forward for further participation in differentiation. As they are in movement, the host cells participate in the formation of new embryo. It creates organs that normally never would have formed before. In the secondary embryo, a somite could be seen containing both pigmented (that is, the donor) and the unpigmented (which is the host) tissues. What was more shocking was, the dorsal lip was able to interact strongly with the host tissue to form a fully formed neural tube derived solely from the host’s ectoderm. Back then, Spemann referred to the dorsal lip cells and their derivatives as the organizer. The reason being was because-

  • They could induce the host’s ventral tissue to change their fates to form a neural tube and a dorsal mesodermal tissue (most commonly the somite).
  • They could systematically organize host and donor tissues into a secondary embryo with clear anterior-posterior and dorsal-ventral regions.

Because, there are numerous inductions during the embryonic developments, this key induction wherein the progeny of dorsal lip cells induces the dorsal axis and the neural tube is traditionally called the Primary embryonic organizer.

REFERENCES-

https://www.khanacademy.org/science/biology/developmental-biology/signaling-and-transcription-factors-in-development/a/frog-development-examples

https://en.wikipedia.org/wiki/Spemann-Mangold_organizer

AFFINITY CHROMATOGRAPHY

BY: SHAILY SHARMA (MSIWM041)

Chromatography is a technique which is widely used for the separation of mixtures based on the physio-chemical differences between the stationary and the mobile phase of the chromatographic apparatus. The sample to be separated is dissolved in the mobile phase, which passes over the stationary phase (the immobile/fixed surface) and carries out the process of separation of molecules. 
See the source image

Most chromatographic processes employ and rely on these physio-chemical differences between the stationary and mobile phases for the process of separation of molecules. For example, gel permeation chromatography, ion exchange chromatography etc.

However, an exception is affinity chromatography. Unlike gel permeation and ion exchange chromatography, affinity chromatography exploits the tendency or capacity of certain biomolecules to bind specifically and non-covalently to other molecules called ligands (i.e., “bio-specificity of molecules)

PRINCIPLE:

One of the most familiar concepts for anyone involved in the studies of biochemistry is “bio-specificity”. 

Specificity is a molecular recognition mechanism which operates through structural and conformational complementarity between the biomolecule and its substrate. It is known that a given enzyme only bind/react with its specific group of substrates and not with any other. 

This bio-specificity may not always be limited only to enzymes. Other examples of this kind of biomolecular interactions include;

  • A given hormone binds only to its specific glycoprotein (known as the hormone receptor).
  • Antibodies specifically bind to only a given antigen which is shaped/has a confirmation specific to that antibody.See the source image

To understand this principle better with respect to affinity chromatography, let us suppose that an enzyme is to be purified from a mixture of thousands of proteins.

  •  Let the substrate analogue (molecule resembling the substrate but not capable of reaction) specific for this enzyme (which is to be separated) be coupled to the stationary phase (let’s say the stationary phase for this particular example be e.g., agarose). 
  • All other molecules, which have no specificity for the ligand and are incapable of binding will pass down and out of the column, (as seen in the second diagram in the picture.)
  • Lastly, once the other substances are eluted, the bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength, or polarity conditions which would completely alter the strength of binding of the enzyme to the substrate and help in the elution of the desired product.

COMPLICATINS THAT ARISE DURING AFFINITY CHROMATOGRAPHY:

There are some complications which arise during affinity chromatography, they may be due to the nonspecific adsorption of sample components other than the desired one on to the matrix. Usually, when this happens, ionic and hydrophobic interactions are involved. 

This complication may be taken care of by judicious choice of operating conditions (e.g., pH, temperature, or ionic strength) in such a way that the physical conditions exclusively favour the binding of the desired molecule to the substrate and not any other undesired molecule.  

Another type of complication arises when one uses ligands, which interact with more than one macromolecule present in a given mixture.

IMPORTANT VARIALBES INVOLVED:

  1. The type of matrix that is used.
  2. The type of ligand used.
  3. The conditions of binding and elution of the sample from the matrix.

A general discussion of these points has been given below:

Properties of the supporting matrix:

  1. The matrix that is used in the process must be chemically inert to other molecules in order to minimize the rate of non-specific adsorption.
  2. The matrix should possess good flow properties.
  3. The matrix should be able to remain stable even at varying levels of pH and temperature, ionic strengths and denaturing conditions.
  4. It should be highly porous to provide a large surface area for the attachment of ligand.

Ligand Selection:

  1. The ligand used should be capable of forming moderately strong interactions with the desired macromolecule. 
  2. The ligand that is to be bound must possess functional groups that may be modified to form covalent linkages with the supporting matrix. 

Ligand attachment:

  • The covalent coupling of the ligand to the supporting matrix occurs in two steps;
  1. Activation of the matrix functional groups, and
  2. Covalent attachment of the ligand to the matrix.
  • The chemical methods that are used must be relatively mild so as to ensure no/minimal damage to the ligand or the matrix.

APPLICATIONS OF AFFINITY CHROMATOGRAPHY:

  1. Affinity chromatography has been widely used for the purification of varied number of macromolecules which are capable of showing ligand specific interactions like enzymes, hormones, antibodies, nucleic acids, membrane receptors etc.
  2. Affinity chromatography has also been used for the purification of cells as this technique is known to affect the viability of cells less than other chromatographic techniques. Cells that have been purified using this technique include fat cells, T and B lymphocytes, spleen cells, lymph node cells etc.
  3. An extension of affinity chromatographic principles is using magnetic gels. The gel beads contain a magnetic core that is chemically coupled to a protein ligand. The suspended cells are then allowed to interact with the microspheres. When a magnetic field is passed, the cells of interest move towards the poles of the magnets thereby getting separated. The cells can be collected by removing the magnetic field. 

Immunoglobulin negative thymocytes and neuroblastoma cells are separated using this technique.

Sources:

Biophysical Chemistry principles and techniques – Upadhyay and Nath

INTRODUCTION TO VIRUSES

BY: SHAILY SHAMA (MSIWM041)

HISTORY:

  • Throughout the course of modern human history, the source some viral infections such as smallpox, polio, and the Spanish flu have been quite unknown to humans. They have been diseases which have had deadly effects on humanity. All that was known about these diseases was that they spread via person-to-person contact.
  • In the second half of the1800’s, Louis Pasteur postulated that the disease rabies was caused by a “living thing” which is in all probability, smaller than bacteria itself. This postulation of his led him to develop the first vaccine against rabies in 1884. 
  • The initial discovery of the light microscope held some promise with respect to the observation of the agents causing such sever diseases however, it was found that the microscope could only be used to observe bacteria, protozoa and fungi. The size of virus was smaller than these agents and therefore, could not be observed under a microscope.
  • In the 1890’s, D. Ivanovski and M. Beijerinck showed that a disease caused in plants was cause by the tobacco mosaic virus which served as the first properly substantial revelation related to viruses.
  • This discovery was then followed by two other scientists, Friedrich Loeffler and Paul Frosch who isolated the virus that caused foot and mouth disease in cattle. 

POSITION OF VIRUSES IN THE BIOLOGICAL SPECTRUM:

Viruses are extremely unique entities which are capable of infecting almost every single type of cell known including bacteria, algae, fungi, plants, animals and even protozoa. 

Questions regarding the nature of viruses like their origination, their state of existence (alive or non-living), their distinct biological characteristics etc. are still very dominant in the scientific world.

Some ideas that have been addressed are:

  • Viruses are considered to be the most abundant microbes on earth, in terms of number.
  • Viruses are considered to be obligate intracellular parasites that are incapable of dividing or multiplying unless they invade a specific host cell. They multiply by taking over the hosts genetic and metabolic machinery of the cells of the host.
  • They are said to have been in existence for billions of years and have arisen from the loose strands of genetic materials released by the cells. (This is one widely accepted theory. However, it too has faced some criticism.)

PROPERTIES OF VIRUSES:

  1. Viruses are obligate parasites of protozoa, bacteria, fungi, animals, plants, and algae.
  2. They have an ultramicroscopic size range from 20nm up to 450 nm in diameter.
  3. They have a very compact and economical structure. They are not cellular in nature.
  4. They are inactive macromolecules outside the host cell and only get activated inside the host cells.
  5. Viral nucleic acid can be DNA or RNA but never both together.
  6. Their basic structure consists of a protein coat (the capsid) surrounding the nucleic acid core.
  7. They lack the enzyme and machinery for basic metabolic processes and synthesis of proteins.
  8. Virus multiply by taking over the host machinery and genetic material.

THE GENERAL STRUCTURE OF VIRUSES:

SIZE RANGE:

  • Viruses represent the smallest infectious agents in the biological world. (with a few exceptions)
  • They lie within the ultramicroscopic size range with sizes usually less than 0.2 micrometre. They are so small that one requires an electron microscope to detect or examine their structure.
  • The animal size range may vary from parvoviruses (which are around 20 nm in diameter) to megaviruses and pandoraviruses (which are up to 1000nm in width) that may be as big as small bacteria.
  • Some viruses which are cylindrical may be relatively long (around 800nm) but have a very narrow diameter (around 15nm).
  • Negative staining using an opaque salt in combination with electron microscopy can be used for the observational studies of viruses.

STRUCTURAL COMPONENTS OF A VIRUS:

Viruses have a crystalline appearance due the occurrence of regular, repeating molecules. A number of purified viruses even form large aggregates and crystals when subjected to special treatments. 

The general plan of all viruses or the general architecture is quite simple. Almost all viruses contain a protein coat or the capsid which encloses the viral genome which may be a DNA or an RNA sequence. Apart from this, viruses only contain those parts which are needed to invade and take control over the hosts cellular machinery. 

Some important terms are:

  • Capsid: The outer covering or the shell of the virus that surround the central nucleic acid core of the virus.
  • Nucleocapsid: The outer shell along with the nucleic acid core is called the nucleocapsid.
  • Naked viruses: Viruses that do not contain a nucleocapsid layer are called naked viruses.
  • Enveloped viruses: Some virus classes possess an additional covering which is external to the capsid which is called an envelope. This envelope structure is usually a piece of the hosts cell membrane. These types of viruses are called enveloped viruses.

THE VIRAL CAPSID:

The capsid layer of the virus, when magnified immensely, shows the appearance of small, prominent, geographic structures. These structural subunits of the capsid are called capsomeres. 

These capsomeres are capable of self-assembling into the finished capsid structure. Depending on the shape and assembly of the capsomeres, the resulting structure can be of two types; 

  1. Helical: Helical capsids have rod shaped capsomeres that bind together to form a structure similar to hollow discs (like a bracelet). During the process of formation, these discs link together to form a continuous helix.See the source image
  1. Icosahedral capsids: An icosahedron is a three-dimensional, 20-sided figure with 12 evenly spaced corners. Some viruses also show such an arrangement in this shape.
  2. Complex viruses: Such viruses may have a specific head, a neck and other structures specific for the invasion of host cells. The most common examples of such viruses is phage viruses.

THE VIRAL ENVELOPE:

Enveloped viruses, when released from the host cells sometimes carry forward a piece of the hosts cell membrane with them in the form of an envelope. Although it is derived from the host, the envelope is different in the virus because the normal proteins of the host get replaced with the viral proteins. 

VIRAL GENOME:

At the center of the viral structure, within the capsid lies the viral genome which may be single or double stranded. The genetic material may be RNA or it may be DNA but it is never both even in viruses. The genome may be a few hundred to thousands of base pairs long. 

Sources:

Foundations in Microbiology – Talaro, Kathleen P

BIOSAFETY CABINET

BY: Ezhuthachan Mithu Mohanan (MSIWM043)

A biological safety cabinet is an enclosed but ventilated workspace. It is mostly used while working with contaminated pathogens. There are many levels in BSC, depending on the contaminants. There are mainly three states of protections

  1. Personal protection
  2. Product protection
  3. Environment protection

There are three classes for BSC based on containment capabilities

  • Class 1 cabinet: This is used to provide personal as well as environment protection. Biological agents should be of low to moderate risk. BSL 1,2 and 3
  • Class 2 cabinets: This is used to provide personnel, environment as well as product protection. Biological agents should be of low to moderate risk. BSL 1,2 and 3
  • Class 3 cabinet: Also known as glove box. This is used to provide personnel, environment as well as product protection .BSL 4(highly infectious agents).

Biosafety level:


BSL are standard level defined by Biosafety in Biomedical Laboratories (BMBL), Which mainly measures the protection needed in a laboratory setting to protect workers, environment and public. Biological risk assessment (BRA) is used to conduct each experimental protocol. BRA are assessments which mainly used to evaluate the following The infectious or toxins transmitted and can cause disease.Availability of medical treatmentsHealth checkup and training of lab employees

The main requirement for any given Biosafety level are Laboratory designPPE (Personnel protective equipment)Biosafety equipment

There are mainly 4 BSL

Biosafety Level 1: BSL1 is used for those infectious agents which is mainly not considered for causing health risk to healthy individuals. The procedures followed are mainly under the category of Standard Microbiological practices (SMP). There comes no specific requirement of special equipment or design features. The main necessities are cleaned surfaces, withstanding basic chemicals etc.

Biosafety Level 2: BSL2 focus on study of moderate risk infectious agents. The risk of getting infectious may be due to accidental inhalation, swallowing, exposed to cut skin etc. The specific requirement such as hand washing sinks, eye washing, automatic door and lock.  There is basic requirement of equipment which can decontaminate lab waste, incinerator, and autoclave.

Biosafety Level 3:  BSL 3 mainly focus on infectious agents which can be potentially lethal, when accidentally inhaled or exposed. For any research studies with biological agents whose spread can be lethal, BSL 3 is used. The controlled airflow or sealed enclosures are important for such agents. Easy decontamination, directional airflow, two self-closing or interlocked, doors, sealed windows, properly sealed wall surfaces, filtered ventilation etc. are basic necessities under the category of BSL2. There is basic requirement of equipment which can decontaminate lab waste, incinerator, and autoclave.


Biosafety Level 4: BSL 4 mainly focus on those agents which have high risk of aerosol transmission an may be life threatening disease, having no vaccines or therapy developed till date. It includes all BSL 3 features, along with it should be developed in an isolated zone. Significant training should be provided for the workers. Careful and controlled access. There are mainly two types of BSL4 

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Pathogen profile of Klebsiella pneumoniae:

BY: SHAILY SHARMA (MSIWM041)

Klebsiella pneumoniae is the pathogen that causes pneumonia and septicemia (blood infections) which is usually found in the normal flora of the mouth skin and the intestines. It may also cause meningitis and bacteremia.

Normally when they occur in the intestines, the organism is harmless. However, the spreading of the organism to other parts pf the body and under certain conditions causes diseases in humans.

Clinically, it is one of the most significant members of the genus Klebsiella of the Enterobacteriaceae. In the recent years, one of the most important pathogens of the nosocomial infections, involving the urinary and the pulmonary systems, has been the Klebsiella species. 

Naturally, it occurs in the soil and about 30 percent of the strains of Klebsiella show nitrogen fixing abilities under anaerobic conditions.

  • Microscopic morphology: 
  • It is a gram-negative organism that occurs in the encapsulated form.See the source image
  • It is a straight rod-shaped organism around 1 to 2 micrometers in length.
  • It is a non-motile organism which is facultatively anaerobic.
  • On MacConkey agar medium it appears as a mucoid lactose fermenter.
  • Habitat:
  • It is usually found in the mouth, skin and intestines of humans as normal flora.
  • Virulence factors:
  • The bacterium possesses a thick polysaccharide capsule which prevents the ingestion of the organism by the hosts phagocytes and somatic antigen from being detected by the host’s antibodies.
  • The bacterium also shows the presence of a thick lipopolysaccharide capsule which makes the serum complement activation more difficult for the host’s immune system.
  • K. pneumoniae protects itself and avoids damage by the host’s complement proteins by the extreme length of the molecules comprising the capsule and allows the membrane attack complex (MAC) to form away from the membrane. This helps in the prevention of opsonization and insertion of the MAC.
  • The bacterium uses the host’s ferric-siderophore receptors to activate its own Enterobactin-mediated iron-sequestering system.
  • Primary infections/disease:
  • Most commonly, Klebsiella causes pneumonia. Typically, in the form of bronchopneumonia and bronchitis. 
  • The organism is transmitted when a person is directly exposed to the bacteria. The bacteria must enter either directly enter the respiratory tract to cause pneumoniae or the blood stream to cause a bloodstream infection.See the source image
  • These patients have a higher tendency to develop other complications like lung abscess, cavitation, empyema and pleural adhesions.
  • Apart from or in addition to pneumonia, Klebsiella causes other infections like infections of the lower biliary tract, urinary tract and also infection of and around surgical wound sites. 
  • The range of these clinical infections include diseases like cholecystitis, diarrhea, upper respiratory tract infection, meningitis, sepsis etc. 
  • The bacterium can also enter the blood post sepsis and septic shock.
  • In most cases, patients suffering from Klebsiella pneumoniae cough up a characteristic sputum in addition to fever, nausea, tachycardia, and vomiting.
  • If a person acquires the infection in a community setting, like in a mall, community-acquired pneumonia occurs.
  • A urinary tract infection (UTI) may also be caused by the pathogen if it enters one’s urinary tract. It typically occurs in older women.
  • The pathogen may also cause wound infections like cellulitis, necrotizing fasciitis and myositis if it enters through a break in the skin and affects the soft tissue.
  • Diagnosis:
  • Susceptibility testing for (ESBL) Extended spectrum β-Lactamase.See the source image
  • Other tests that can be done for the diagnosis of K. pneumoniae include:
  1. CBC or complete blood count.
  2. Sputum culturing of the patient.
  3. Radiography of the chest to check for lung abnormalities visually.
  4. CT scans.
  • Treatment:
  • The treatment for Klebsiella pneumoniae infections mainly depends upon the patient’s health conditions, medical history and the level of severity of the disease. 
  • Treatment is by antibiotics like aminoglycosides and cephalosporins.

Sources: 

 Ryan, KJ; Ray, CG, eds. (2004). Sherris Medical Microbiology (4th ed.). McGraw HillISBN 978-0-8385-8529-0.

 “Klebsiella species – GOV.UK”. www.gov.uk

Human Mitochondrial DNA

BY: Ezhuthachan Mithu Mohanan (MIWM043)

DNA within mitochondria was first detected in 1963. Human mitochondria represent the mammalian mtDNA. Human mtDNA is 16,569 bp, double stranded and circular. It codes for 13 polypeptides, belonging to OXPHOS family. mtDNA also codes for 22 tRNA,2 rRNA. It also has control noncoding regions. Various nuclear coded factors also known as precursor polypeptides are essential for the expression and maintenance of mtDNA. It was Sanger who found that circular mtDNA in vertebrates, has both Light Strand and Heavy strand. There are many differences considering nuclear DNA and mitochondrial DNA, which are as follows 

MITOCHONDRIAL DNA NUCLEAR DNA 
Present in mitochondria Present in nucleous
One cell contains 0.25% mtDNAOne cell has 99.75% n DNA
Mutation rate of mtDNA is 20 times faster than nDNASlow mutation rate 
circular Linear
1000’s of mtDNA copies/ cell2 Copies / cell
HaploidDiploid
Maternally inherited Biparental inheritance
Replication repair mechanism is absent Replication repair mechanism is present
Reference sequence by Anderson  and Colleagues in 1981Reference sequence in Human Genome Project in 2001
Size of genome is 16,569 bp Size of genome is 3.2 billion base pair

The genetic code of nuclear DNA differ from mtDNA as such ‘TGA’ codes for tryptophan in vertebrate mitochondria, while it is a stop codon for nuclear DNA. ‘ATA’ codes for Isoleucine in cytosol, while it codes for methionine in mitochondria.

Mitochondrial Inheritance: 

With most of the evidence provided, mostly there is maternal inheritance of mitochondria. Due to nucleotide imbalance and reduction in fidelity of polymerase γ, it causes higher mutation rate. This can be used as approach for human identity test, studying evolutionary and migration pattern.

mtDNA replication:  

Factors for mtDNA  replication: 

DNA Polymerase γ is the polymerase enzyme, it is a heterotrimer with one catalytic subunit ( POLγA). POLγA has 3’-5’ exonuclease activity for proofreading. TWINKLE is DNA Helicase which unwinds double stranded DNA. mtSSB is binds with single stranded DNA to protect from nucleases. Vinograd and coworkers proposed the strand displacement theory, which emphasize continuous DNA synthesis on H and O strand. The replication initiation begins from OH Strand, which proceeds unidirectional. During OL replication stem loop structure is formed which block mtSSB from binding, initiating primer synthesis. Thus the two strand synthesis occur in a continuous manner , until two complete double stranded DNA is formed.A triple-stranded displacement loop structure also known as D Loop is formed, When 7S DNA remains bound to parental L strand, while parental H-Strand is displaced.  The role of mtDNA D loop is not completely understood. 

Mitochondrial Diseases:

A dysfunction in mitochondria leads to mitochondrial disorder. Heteroplasmy  is condition due to presence of mutant mitochondrial DNA .  Various Mitochondrial disorders are as follows:

  1. Mitochondrial Myopathy:  Presence of ragged red muscle fibres is due to accumulation glycogen and neutral lipids which leads to decreased reactivity of cytochrome c oxidase
  2. Leber’s hereditary optic neuropathy : This is maternally inherited, which causes degeneration of retinal ganglion cells
  3. Leigh syndrome: It is a neurometabolic disorder affecting CNS( central nervous system)
  4. Myoneurogenic gastrointestinal encephalopathy : Autosomal recessive disorder. It is due to mutation of TYMP gene
  5. Mitochondrial DNA depletion syndrome: It is also known as Aplers disease. This is caused my mutation inTK2 gene.

GEL PERMEATION CHROMATOGRAPHY

BY: SHAILY SHARMA (MSIWM041)

Chromatography, broadly, is a technique to separate two phases which are mutually immiscible. The phases are brought in contact with each other and one of these phases is stationary while the other phase is a mobile phase which moves over the surface of the stationary phase or percolates it. The interactions of the sample mixture which are established after the movement of the mobile phase over the stationary phase lead to the separation of the desired compound present in the mobile phase based on the differences in the physio-chemical properties of the two mediums.

There are various techniques used in the process of chromatography like plane chromatography, paper chromatography, thin layer chromatography, column chromatography etc. 

Based on these techniques, further different types of chromatographic procedures have been curated which include adsorption chromatography, partition chromatography, gel permeation chromatography, ion exchange chromatography and affinity chromatography. 

GEL PERMEATION CHROMATOGRAPHY:

Gel permeation chromatography is a chromatographic procedure that separates molecules based on their molecular size. This method has various other names such as molecular sieve, gel filtration and molecular exclusion chromatography.

This method is widely used due to its many advantages, some of which are:

  1. It is a very gentle technique that permits the separation of labile molecules.
  2. It is a technique in which the solute recovery is almost 100%.
  3. The reproducibility of this technique is high.
  4. The technique is not very time consuming and is relatively inexpensive.

PRINCIPLE: 

The principle of gel permeation chromatography is relatively very simple.

A long column filled with gel beads or porous glass granules is allowed to attain equilibrium with a solvent which is suitable for the separation of the desired compound or molecule. 

Assuming that the mixture to be separated contains molecules of varying sizes, when this mixture will be passed through the column containing the beads;

  • The larger molecules pass through the interstitial spaces between the beads and do not enter the spaces inside the beads. This occurs because the diameter of the molecule is larger than the pore size of the beads.
  • Therefore, the larger molecules are able to pass down the column with little or no resistance.
  • The small molecules however, have a size which is smaller than the diameter of the pores of the beads and therefore these molecules enter the beads and reach the end of the column after a longer period of time due to the resistance that is created by the passage of the molecules through the beads.See the source image

The degree of retardation (or the extent of the time taken by the molecule to reach the bottom) is proportional to the time it spends inside the pores of the gel which is a function of the molecules pore size and the molecules size.

The molecules which have a diameter equal to or smaller than the pore size, do not enter and are said to be excluded. Therefore, the exclusion limit of a gel can be defined as the molecular weight of the smallest molecule which is not capable of entering the pores. For e.g. linear polysaccharides and fibrous proteins have a much lower exclusion limit as compared to the globular proteins of comparable molecular weight. 

TYPES OF GELS USED:

A good gel filtration medium should possess a few properties or characteristics like:

  • The material of the gel should be chemically inert.
  • It should contain a small number of ionic groups.
  • The material of the gel should have a wide variety of particle and pore sizes.
  • It should have a uniform particle and pore size.
  • The material of the gel should be of high mechanical rigidity.

Some examples of the types of gels used in gel permeation chromatography include sephadex, polyacrylamide, agarose, styragel etc.

TECHNIQUE/PROCESS OF GEL PERMEATION CHROMATOGRAPHY:

This chromatographic technique can be performed wither by column or by thin layer chromatography techniques. 

The initial step of the process is:

  1. Preparation of the beads:

Prior to use, the gels used in the column must be converted to their swollen form by either soaking them in water or using a weak salt solution. The greater is the porosity of the gel beads, more will be the time taken for them to attain equilibrium and reach their maximum size.

If porous glass granules are used, the gel beads need not be hydrated at all. 

  1. Preparation of the column:

The gel beds, in their swollen form, are mounted or supported on a column on a glass wool plug or nylon net and the previously swollen gel is added in the form of slurry. The preparation is then allowed to settle. Air bubbles must be removed by connecting the column to a vacuum pump. It must be noted that the level of the liquid must not go lower than the top of the bed. See the source image

  1. Addition of the sample:

The sample must be added from the top of the column using a funnel and the volume of the sample that must be added depends on the size and the type of the gel that is used in the preparation of the stationary phase. 

  1. Collection of the sample:

The eluant used is steadily added and the effluent can be collected in various various fractions in separate tubes.

  1. Detection of sample: 

The most common detection methods include the collection and analysis of the fraction and continuous methods in which the UV absorption, refractive index or radioactivity is measured.

APPLICATIONS:

  1. Gel permeation chromatography is mainly and widely used for the separation of biomolecules and purification. Biomolecules like proteins, hormones, enzymes etc. can be separated using this technique.
  2. This technique is especially useful for the separation of 4S and 5S tRNA.
  3. This technique is used for the separation of salts and small molecules from macromolecules.
  4. Gel permeation chromatography is also chiefly used for the detection of molecular weights of macromolecules. 

Sources:

Biophysical Chemistry Principles and Techniques by Updhyay and Nath

ION EXCHANGE CHROMATOGRAPHY

BY: SHAILY SHARMA (MSIWM041)

Chromatography is a separation and purification technique which is used for the separation of solutes in a mixture, biomolecules etc. on the basis of distribution of the sample to be separated between stationary phase (phase which is not mobile and is usually mounted against a support like a chromatographic column) and the mobile phase (which is continuous/is poured or passed over the stationary phase)

Ion Exchange Chromatography is a method used for the separation as well as purification of ionically charged biomolecules like proteins, polynucleotides, nucleic acids etc. 

This technique finds a wide array of applications in the scientific world because of its simplicity and high resolution. 

  • PRINCIPLE:

The process of ion exchange can be defined as the reversible exchange of the ions present in a solution, with the ions electrostatically bound to the inert support medium.

The main factor which governs the process of ion exchange chromatography is the electrostatic force of attraction present between the ions. This electrostatic force of the ions depends on their relative charge, radius of the hydrated ions and the degree of non-bonding

interactions.

Usually, ion exchange separations are carried out in columns packed with an ion-exchanger. The ion-exchanger is a support medium which is inert and insoluble. The medium may be capable of covalently binding to positive (anion exchanger) or negative (cation exchanger) functional groups. The ions which do bind to the exchanger electrostatically are called counterions. 

The conditions of separation can be manipulated in such a way that some compounds are electrostatically bound to the ion exchanger while some are not, therefore, helping in the separation of the desired compound.

The sample which contains the sample to be separated is allowed to percolate through the exchanger for a certain amount of time that will be sufficient for the equilibrium of the ions to be achieved. 

E–   Y  E–   X+ + Y+

In the equation mentioned above, 

  • E–  : Charged cation exchanger.
  • Y+ : Counterion of the opposite charge associated with the exchanger matrix.
  • X+ : Charged molecule bearing a charge similar to the counterion to be separated. This molecule is capable of exchanging sites with the counterion as shown above.

Once the exchange of the counterion with the sample has been achieved, the rest of the uncharged and like charged species is washed out of the column.

The ions that did bind can then be eluted out by either percolating the medium with increasing concentration of Y+ (works by increasing the possibility that the Y+ will replace the X+ in the above-mentioned equation due to it being present in a higher concentration). The elution can also be carried out by increasing the pH of the solvent and hence converting X+ to an uncharged species.

Simply put, once the sample containing the specific ions to be separated it passed through the ion exchanger column, the sample ions (which act as counterions to the ions of the exchanger column) bind with the ions on the exchanger column and form associations. However, the ions of the same charge as the exchanger column, present in the sample solution, repel each other and, therefore, do not bind and pass through the column.

The principles which have been mentioned above also apply to other macromolecules such as proteins and nucleic acids which are capable of showing the presence of both positive and negative ions. The type of molecules can bind to both anionic and cationic exchangers. 

  • TYPES OF ION EXCHANGE RESINS:

The two main types of materials used to prepare ion exchange resins are:

  1. Polystyrene, and
  2. Cellulose

Polystyrene resins are prepared by the polymerization reaction of styrene and divinyl benzene. These resins are very useful for separating compounds with a small molecular weight. 

Cellulose based resins have a much greater permeability to macromolecular polyelectrolytes as compared to polystyrene resins and they also possess a much lowers charge density. 

Based on the type of charge carried by these ion exchangers and their strength, the ion exchange resins can broadly be classified into four types:

Strong cationic exchange resins:Weak cationic exchange resins:
Sulphonated polystyreneSulphopropyl cellulose Condensed acrylic acidCarboxymethylcellulose
Strong anionic exchange resins:Weak anionic exchange resins:
Polystyrene with -CH2NMe3ClDiethyl (2 hydroxypropyl)quaternary amino celluloseDiethylaminoethyl celluloseDiethylaminoethyl agarose
  • CHOICE OF BUFFERS:

The buffer is that component of the chromatographic column that helps in the maintenance of the pH. The choice of these buffers is usually dictated by the compounds to be separated and whether the ion exchange is anionic or cationic.

  • Anion exchange chromatography should be carried out with cationic buffers.
  • Similarly, cation exchange chromatography should mostly be carried out with only anionic buffers for satisfactory separation and results. 

      Examples of some buffers used in this technique are:

  • Ammonium acetate
  • Ammonium formate
  • Pyridinium formate
  • Ammonium carbonate etc.
  • APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY:
  1. The most significant application of ion exchange chromatography is in amino acid analysis.
  2. This technique is used to determine the base composition of nucleic acids.
  3. Ion exchange chromatography is used as a method of purification of water. Water is completely deionized using this technique.
  4. This technique is used for the ultra-purification of metal ion free reagents.
  5. It can also be used for the separation of a varied number of vitamins, biological amines, organic acids as well as bases. 

Source:

Biophysical Chemistry principles and techniques – Upadhyay and Nath

Overview of infections of respiratory tract and its pathogens

By: Shaily Sharma (MSIWM041)

  • A brief overview of the infections of the respiratory tract and its pathogens:

The respiratory tract along with the gastrointestinal tracts is one of the major connections between the interiors of the body and the outside environment.

The respiratory tract is the pathway is that pathway of the body through which fresh oxygen enters the body and removes the excess carbon dioxide which is not needed by the body. 

  • Anatomy of the respiratory system:
  • Broadly, the respiratory system of humans can broadly be divided into two distinct areas; the upper and the lower respiratory tracts.
  • The parts that consist the lower respiratory tract are:
  1. Trachea
  2. Bronchi, and
  3. Bronchioles
  • The respiratory pathway begins with the nasal and the oral passages. These passages serve to humidify the air that is inspired. These pathways extend past the nasopharynx and the oropharynx to the trachea and then to the lungs.
  • The trachea is the organ that divides into the bronchi, which then further subdivides into the bronchioles. The bronchioles are the smallest branches of the trachea which finally terminate into the alveoli.
  • Approximately 300 million alveoli are said to present in the lungs. These mainly serve as the primary, microscopic, gas exchange structures of the respiratory tract.

See the source image

  • The lungs (along with the respiratory system) and the heart lie in the thoracic cavity. 
  • The thoracic cavity has three partitions that are separated from one other by the pleura (the pleura majorly cushions the lungs and reduce the friction which may develop between the lungs, rib cage and the chest cavity. It is a two layered membrane covering the lungs.)
  • The lungs occupy the right and the left pleural cavity while the mediastinum (the space between the right and the left lungs) is occupied by the esophagus, trachea, large blood vessels along with the heart.
  • Pathogenesis of the respiratory tract:
  • The success of an organism to cause disease is mainly dependent on the organism’s ability to cause disease (pathogenesis), and
  • The human hosts ability to prevent the infection (strength of the host’s immune system)
  • The host factors that help in non-specifically protect the respiratory tract from infection are:
  1. Nasal hair
  2. Convoluted passages and the mucous lining of the nasal turbinate
  3. Secretory IgA and non-specific antibacterial substances (like lysozyme) in respiratory secretions
  4. The cilia and the mucous lining of the trachea and reflexes such as coughing and sneezing. 
  • In addition to the non-specific hosts defenses, normal flora of the nasopharynx and the oropharynx help in the prevention of colonization of the upper respiratory tract. 

Microorganism factors:

Organisms possess certain traits that promote colonization leading to infection in the host. The factors that influence the respiratory tract infections are –

  1. Adherence: 
  • The potential of a microorganism depends, in one way or the other, on its ability to establish a stable contact/foothold on the surface of the host by the process of adherence. 
  • The ability of microorganisms to adhere to the host surface is dependent on two factors:
  1. Presence of normal flora, and
  2. Overall state of the host.
  •  Surviving or growing on host tissue without causing harmful effects is called colonization. 
  • Most etiologic agents must first adhere to the mucosa of the respiratory tract to some extent before they can cause harm.
  • Example: Streptococcus pyogenes possess specific adherence factors and its gram-positive cell wall contains lipoteichoic acids and M proteins. Many gram-negative bacteria like Enterobacteriaceae, Pseudomonas spp., Bordetella pertussis, adhere by the means of proteinaceous fingerlike projections called fimbriae. 
  • Viruses possess either a hemagglutinin or other proteins that mediate that epithelial attachment.
  1. Toxins
  • Certain microorganisms are considered to be etiologic agents of disease because they possess virulence factors that are expressed in every host. 
  • Example: Corynebacterium diphtheriae. 
  • Some strains of Pseudomonas aeruginosa also produce toxins which are similar to the toxins of Diphtheria.
  •  Bordetella pertussis which is the causative agent of whooping cough produces toxins that play a role in inhibiting the activity of phagocytic cells and damaging the cells of the respiratory tract.
  1. Microorganism growth
  • Pathogens cause disease by merely growing in the host tissue, interfering with normal tissue function and attracting host immune effectors, such as neutrophils and macrophages.
  • Example: S. pyogenes, M. tuberculosis, Mycoplasma pneumoniae, etc.
  1. Avoiding the Host Response
  • Certain respiratory tract pathogens possess the ability to evade host defense mechanisms.
  •  S. pneumoniae, H. influenza, K. pneumoniae and others possess polysaccharide capsules that serve both to prevent engulfment by phagocytic host cells and to protect somatic antigens from being exposed to host immunoglobulins.
  • Organisms of the respiratory tract and agents that cause diseases: 

Pathogens may or may not cause the respiratory infection but can be present as a part of normal flora.

  •  Some of the pathogens that exist and results in the respiratory infection are referred to as true pathogens. 
  •  Some of the pathogens that are present in the body but never cause an infection until and unless they are met with the favorable conditions are called

as opportunistic pathogens.  

  • Possible pathogen: they are the pathogens that are likely to cause respiratory

infections.

  • Example: Actinomyces spp., Haemophilus influenzae, Enterobacteriaceae, etc.See the source image
  •  Rare pathogen: pathogens that may cause a respiratory infection are rare

pathogens. Example: Coxiella burnetti, Brucella spp., Salmonella spp, etc.  

  •  Definite respiratory pathogen: pathogens that always cause respiratory infections are called as definite respiratory pathogens.
  • Example: Bordetella pertussis, Blastomyces dermatitidis, Legionella spp., etc.
  • Different types of agents that cause respiratory diseases are bacteria, fungi or

viruses.  

  •  Bacterial agents: the bacterial agents that cause respiratory infections are

Mycoplasma spp., Streptococcus pneumoniae and Neisseria meningitides. 

  • Fungal agents: the fungal agents that cause respiratory infections are Candida

albicans, Cryptococcus neoformans and Histoplasma capsulatum.  

  •  Viral agents: the viral agents that cause respiratory infections are human

metapneumovirus, adenovirus, enteroviruses, and herpes simplex virus. 

  • Major respiratory diseases are caused by M. tuberculosis, S. pyogenes and

K.pneumonia.

Sources:

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