Differentiate between COP I TRANSPORT and COP II TRANSPORT

COP I TRANSPORT :

1. COP I is a protein complex that coats vesicles transporting protein from the cis end of the Golgi complex back to Rough Endoplasmic Reticulum (RER).
2. This type of transport is retrograde.
3. Initiates budding process on cis - end of Golgi complex.
4. Consists of large protein subcomplexes that are made of 7 different protein sub units : Alpha, Beta, Beta', Gamma, Delta, Epsilon and Tau
5. GTPase activity is carried out by ADP Ribosylation Factor (ARF) during the budding process.

COP II TRANSPORT

1. COP II is a protein complex that coats vesicles transporting proteins from the RER to Golgi apparatus.
2. This type of transport is anterograde.
3. Initiates budding process on Rough Endoplasmic Reticulum.
4. Made of 4 different protein subunits. 2 proteins Heterodimers from the coal complex  are -
Sec 23p/ Sec 24p and Sec 13p/ Sec 31p
5. GTPase activity is carried out by GTPase Sar Ip during the budding process.   

Differences between class 1, class 2 and class 3 RNR (Ribonucleotide Reductase)

CLASS 1 RNR:

1. Present in prokaryotes and eukaryotes.
2. Substrate - NDP (Nucleoside diphosphate)
3. Cofactor = Ox bridged binuclear iron center
4. Reductant- Glutaredoxin  and thioredoxin
5. Conditions = functional under aerobic conditions, requires O2 for activation
6. Method of generation of free radicals = the binuclear Fe3+ center interacts with Tyr122 to form the tyrosyl free radical resulting in the generation of a thiol radical (free radical of cys)
7. Exists as alpha2beta2 or alpha2beta6 oligomers.

CLASS 2 RNR:

1. Present in prokaryotes.
2. Substrate = NDP
3. Cofactor= 5-deoxy adenosyl cobalamin
4. Reductant = Glutaredoxin and thioredoxin
5. It is oxygen independent. Can function under both conditions.
6. Radical is generated by homolytic cleavage of the 5-deoxy-adenosyl-cobalamin cofactor. C-Co (III) bond generating a 5-deoxy-adenosyl radical - used to generate thiol radical.
7. Exists only in alpha2beta2 state.

CLASS 3 RNR:

1. Present in anaerobic prokaryotes.
2. Substrate = NTP (nucleoside triphosphate) 
3. Cofactor = [4Fe- 4S] cluster and requires SAM and NADPH for activity
4. Reductant = Provided by oxidation of formate to CO2
5. It is only functional under anaerobic conditions and sensitive to O2.
6. Generated by NADPH supplied and Fe-S cluster mediated one electron reductive cleavage of SAM to yield the 5-deoxy-adenosyl radical which then generates a stable glycyl radical (free radical of glycine , O2 sensitive radical).
7. exists in alpha2 + beta2

Differences between solid-state and submerged fermentation:

SOLID STATE FERMENTATION:

1. WATER ACTIVITY = less
2. NATURE OF SUBSTRATE = insoluble (molasses)
3. GROWTH = Solid state microbes adhere at surface and grow on it.
4. SUBSTRATE = Agricultural waste products are usually used (wheat, gram)
5. MEASUREMENT AND CONTROL OF PARAMETERS = pH, aeration, temperature, agitation and foaming are difficult to measure and control.
6. FLUCTATION IN RESULT = Result vary from batch to batch
7. FERMENTATION TYPE = Only batch fermentation
8. FOAMING = Not a major problem.
9. TYPES OF PRODUCTS = Extracellular product
10. PRODUCT YIELD = Higher concentration of products due to larger amount of substrate available.
11. DOWNSTREAM PROCESSING = Difficult and tedious.
12. STEARILIZATION = It may take long time.
13. EXAMPLES = Harvested product compositing, ripening of cheese, mushroom cultivation, enzyme production etc.

SUBMERGED FERMENTATION:

1. WATER ACTIVITY = more (main component)
2. NATURE OF SUBSTRATE = soluble
3. GROWTH = microbes grow in the suspended form of fungi form mat on the surface
4. SUBSTRATE = make use of crude or synthetic substrates. (crude cheese- whey, molasses)(lactose ,starch growth etc)
5. MEASUREMENT AND CONTROL OF PARAMETERS = pH, aeration, temperature, agitation and foaming are easy to measure and control.
6. FLUCTATION IN RESULT = Result do not vary much from batch to batch
7. FERMENTATION TYPE = Run on batch, fed-batch or continuous 
8. FOAMING =  a major problem.
9. TYPES OF PRODUCTS = can be used for both intracellular and Extracellular product
10. PRODUCT YIELD = Yield is low as the product concentration gets diluted due to large volume of medium used.
11. DOWNSTREAM PROCESSING = Easy recovery of the product
12. STEARILIZATION = It is easy to sterilize. Autoclaving may also be used
13. EXAMPLES = Vitamins production, Amino acids, ethanol, enzyme production.

Differences between continuous and fed-batch

CONTINUOUS:

1. Nutrient addition - Continuously or intermediary with corresponding withdrawal of same volume of media.
2. Type of system - Open system
3. Growth state - Steady state is maintained
4.  specific growth rate = dilution rate 
5. Cell maintenance - It is in log phase.
6. Application - primary metabolites cannot be used for secondary metabolites.
7. catabolite repression - cannot overcome.
8. Types - chemostat, turbidostatic.

FED-BATCH:

1. Nutrient addition - No withdrawal but nutrients or precursors are added without harvesting the medium 
2. Type of system - Partially closed
3. Growth state - A Quasi (pseudo) state is maintained
4.  specific growth rate may not be equal to dilution rate 
5. Cell maintenance - Not necessarily in log phase.
6. Application - Can be used for both primary metabolites and secondary metabolites.
7. catabolite repression - can be overcome.
8. Types - Fixed, variable cyclic

Diauxic growth curve effect of E.coli

Many enzymes are involved in basic cellular housekeeping functions and are synthesized at a more or less constant rate. These are called constitutive enzymes. Other enzymes are synthesized at rates that vary with the cells circumstances and are termed as adaptative or inducible enzymes.

Lactose metabolizing enzymes are inducible:

Bacteria adapt to their environment by producing enzymes that metabolize certain nutrients e.g., lactose, only when these substances are available. E.coli growing in the absence of lactose are initially unable to metabolize this disaccharide to do so, they require the presence of two-protein- beta galactosidase which catalyses the hydrolysis of lactose to its component- monosaccharide and galactosidase permease or lactose permease which transport lactose into the cell. Lactose or one of its metabolic products most somehow trigger the synthesis of above proteins. Such a substance is known as an inducer. The physiological inducer of the lactose system is 1,6 allolactose but isopropyl thiogalactosidase (IPTG) is also a potent inducer which structurally resemble allolactose-lactose system inducers also stimulate the synthesis of thiogalactosidase-trans-acetylase, an enzyme that transfer an acetyl group from acetyl CoA to the 6-OH group of isopropyl-thiogalactosidase such as IPTG.

Lac system genes form an operon- LAC OPERON

The genes specifying wild type beta-galactosidase, lactose permease and thiogalactosidase transacetylase are designated as z, y and a respectively. These are called lac structural genes (genes that specify polypeptide) and are contiguously arranged on E.coli chromosome.

These genes together with the control element 'p and o' form a genetic unit called an operon. Specifically the lac operon. In the absence of inducer, the lac repressor i.e., the regulatory gene prgene product specifically binds tightly to the O gene so as to prevent the transcription of mRNA. On binding inducer, the repressor dissociates from the operator thereby permitting the transcription and subsequent translation of lac enzyme.

Catabolite repression

Glucose is E.coli metabolite of choice the availability of adequate amount of glucose prevents the full expression of genes that encodes protein involved in the fermentation of numerous other catabolites, including lactose, arabinose, galactose, even when these metabolites are present in high concentration. This phenomenon is known as "catabolite repression" which prevents the wasteful duplication of energy producing energy system.

If E.coli grows in a medium containing both glucose and lactose, it uses glucose preferentially until the sugar is exhausted. Then after a short lag, growth resumes with lactose as the carbon source, this biphasic growth pattern or response is called 'diauxic growth'.

cAMP signals the lack of glucose

The greatly diminished level of cAMP in the presence of glucose is the indication that of mechanism of catabolite repression. The increase in cAMP may be due to the effect of the phosphoenolpyruvate phospho-transferase system (PTS) on the adenyl activity, the enzyme that synthesizes cAMP. Enzyme III of PTS denotes a phosphate to glucose during its transport, therefore it enters the cell as glucose-6 phosphate. The phosphorylated form of enzyme III also activates adenyl cyclase.

If glucose is being rapidly transported by PTS the amount of phosphorylated enzyme III is low and the adenyl cyclase is less active, so the cAMP level drops. At least one other mechanism is involved in diauxic growth. When the PTS is actively transported glucose into the cell, non-phosphorylated enz III is more prevalent non-phosphorylated enz III binds to the lactose permease and allosterically inhibits it, thus blocking lactose uptake.

CAP-cAMP complex stimulates transcription of catabolite repressed operons:

CAP is homodimer of 210 residue subunits that undergo a large conformational change on binding cAMP. This CAP is synonymously called as catabolite gene activator protein or cAMP Receptor Protein (CRP).

CAP-(cAMP)2 complex but not CAP itself binds to lac-operon and stimulates transcription from its otherwise low efficiency promoter in the absence of lac repressor. CAP is therefore a positive regulator in contrast to the lac repressor which is a negative regulator (twins off transcription).

Differnces between batch and continuous

BATCH :

1. It is a closed system.
2. All four phases of microbial growth is present.
3. Primary and secondary metabolite are produced.
4. Nutrients are added only one not in between the fermentation process.
5. Process is stopped after the product is formed.
6. Environment conditions inside the batch culture are not constant.

CONTINUOUS:

1. It is an open system.
2. Only lag or exponential phase is present.
3. No secondary metabolite production.
4. Nutrients are added in between the process.
5. Process is not stopped and product is removed.
6. Environment conditions are maintained at constant rate. 

Differences between primary and secondary metabolite:

PRIMARY METABOLITE:

1. Required for the growth and maintenance of cellular functions.
2. Starting substrates of a medium.
3. Consist of vitamins, amino acids, nucleotides etc.
4. Necessary at log phase of microbial growth.
5. Produced to perform physiological functions and support in development of cell.
6. Same in every species means they produce same product.
7. Produced in large quantities.

SECONDARY METABOLITE:

1. Not required for growth.
2. End products of primary metabolite.
3. Consist of antibiotics, steroids, toxins etc.
4. Produced during stationary phase of cell growth.
5. Important in ecological and other activities of the cell.
6. Varies in different species.
7. Produced in small quantities.

Differences between batch and fed batch

BATCH :

1. It is a closed system.
2. Nothing is added or removed during fermentation.
3. Not used to overcome catabolite repression.
4. Large size vessels are required due to slow growth rate.
5. Cannot extended the growth rate and the phases.
6. Volume remains constant.
7. Less chances of contamination.
8. Less efficient.
9. Primary and secondary metabolite are produced

FED-BATCH:

1. Semi-closed system.
2. Substances are added in a small dose during productive phase (periodically).
3. Used to overcome catabolite repression.
4. Can be run in a simple bioreactor. No need of specialized instruments.
5. Production phase of process may be extended using cyclic fed-batch cultures.
6. Volume increases.
7. Intermediate chances of contamination.
8. Product yield is more.
9. Secondary metabolites are generally produced (e.g., Penicillin)

Tissue culture

WHAT IS TISSUE CULTURE?

It is a technique of growing cells, tissue, organs or whole organism in vitro on an artificial culture media in an aseptic and controlled condition.

Tissue culture involves the use of small pieces of plant tissue (explants) which are cultured in a nutrient medium under sterile conditions. Non-dividing parenchyma, cells of meristem, vascular cambial tissues and embryonic tissues are at early stage of development and exist in an "undetermined" state which rapidly proliferate and produce callus.

CULTURE MEDIA:

Components of a typical culture media includes:

• Organic nitrogen sources (e.g., glycine, inositol)
• Carbon source (e.g., glucose , sucrose)
• Inorganic micronutrients (e.g., Mn, Cu, Zn, B, Na, Cl, I, S, Mo, Co, Al, Ni)
• Inorganic macronutrients (e.g., Fe, Mg, Ca, K, P, N)
• Vitamins (e.g., Nicotinic acid, pyridoxine, thiamine)
• Plant growth regulators (e.g., Auxin, cytokinin)
• Antibiotics (e.g., Kanamycin)
• If media is semi-solid then a good quality gelling agent (Agar)
• Example - MS media, B5 media 

TYPES OF CULTURE MEDIA:

On the basis of consistency of the media, they are of two types:

1. Solid Media/ Semi-solid media: The usual gelling agent for solid or semisolid medium is agar, a hydro colloid derived from red algae.
2. Liquid media: It is also called as broth and contains only dissolved nutrients in water. Liquid media are used for growth of pure batch cultures for fermentation studies and various other purposes while solidified media can be used widely for isolation of pure cultures for estimating viable microbial population and a variety of other purposes.

PREPARATION OF EXPLANTS:

• Meristems are actively mitotically dividing usually free of viruses infection because they lack vascular tissue which transports viruses.
• The smallest possible meristem explants should be taken in order to avoid infection.
• Heat treatment (34-35C) to meristem explants is commonly used to inactivate the virus to reduce the possibility of infection.
• The explants are first sterilized. Common sterilizing agents are 1-2% sodium hypochlorite, 9-10% calcium hypochlorite (10-40 min., young stems, petioles, roots, fruits) and 70% ethanol (2-5 min.. leaves, seeds)

SEED CULTURE:

Seed culture is the type of tissue culture that is primarily used for plants such as orchids. For this method, explants (tissue from the plant) are obtained from an in-vitro derived plant and introduced in to an artificial environment, where they get to proliferate. In the event that a plant material is used directly for this process, then it has to be sterilized to prevent tissue damage and ensure optimum regeneration.

EMBRYO CULTURE:

Embryo culture is the type of tissue culture that involves the isolation of an embryo from  a given organism for in vitro growth.

Embryo culture may involve the use of a mature or immature embryo. Whereas mature embryos for culture are essentially obtained from ripe seeds, immature embryo (embryo rescue) involves the use of immature embryos from unripe/ hybrid seeds that failed to germinate. In doing so, the embryo is ultimately able to produce a viable plant.

CALLUS CULTURE:

This is the term used to refer to unspecialized, unorganized and a dividing mass of cells. A callus is produced when explants (cells) are cultured in an appropriate medium- a good example of this is the tumor tissue that grows out of the wounds of differentiated tissues/organs.

In practice, callus culture involves the growth of a callus (composed of differentiated and non-differentiated cells), which is the followed by a procedure that induces organ differentiation.

ORGAN CULTURE:

Organ culture is the type of tissue culture that involves isolating an organ for in vitro growth. Here, any organ plant can be used as an explant for the culture process (shoot, root, leaf and flower).

With organ culture, or as in with their various tissue components, the method is used for preserve their structure or functions, which allows the organ to still resemble and retain the characteristics they would have in vivo. Here, new growth (differentiated structures) continues given that the organ retains its physiological features. As such, an organ helps provide information on patterns of growth, differentiation as well as development.

MERISTEM CULTURE:

Cultivation of axillary or apical shoot meristem, particularly of shoot apical meristem, is known as meristem culture.

PROTOPLAST CULTURE:

Protoplasts are naked plant cells without the cell wall, but they possess plasma membrane and all other cellular components. They represent the functional plant cells but for the lack of the barrier, cell wall. Protoplasts of different species can be fused to generate a hybrid and this process is referred to as somatic hybridization (or protoplast fusion).

PLANT REGENERATION FROM CALLUS:

• The relative level of auxin and cytokinin controls the shoot and root formation.
• A callus can be regenerated into a whole plant by simply altering the concentration of growth regulators.
• When auxin/cytokinin ratio is high, only roots are formed.
• When cytokinin/auxin ratio is high, shoots are formed.
• When levels of both hormones are intermediate, it results in completely disorganized callus growth.

SOMATIC HYBRIDIZATION:

Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristic of both, a somatic hybrid.

MICROPROPAGATION:

Micropropagation involves in vitro propagation of the selected genotype and ultimate establishment of the plant in the field or a glasshouse.

There are five stages in micropropagation:

1. Preparation of explant : the plant material for in vitro culture is prepared and explants are obtained which requires less aggressive sterilization.
2. Formation of callus
3. Shoot proliferation: a high cytokinin/auxin ratio induces shoot formation and high auxin/cytokinin ratio induces root formation. After 4-8 weeks, the original explant is transformed into a mass of branched shoots or a cluster of basal shoots. The small shoots or clusters are then re-planted on to a fresh medium (in which the cytokinin level can be increased) to increase shoot multiplication.
4. Shoot elongation and root formation: Adventitious and axillary shoots lack roots in the presence of cytokinin, fresh liquid media added to established cultures (double layer technique). Auxins such as NAA are usually required to induce rooting and activated charcoal can be added to the liquid medium in order to adsorb any residual cytokinin.
5. Transfer to a glasshouse: The micro propagated plantlet must acclimatize to the environment of the glasshouse with appropriate humidity and temperature control. Acclimatization can proceed in vitro with bottom cooling, reducing the relative humidity in the head space of the container. The culture vessels are uncapped and placed in the glasshouse several days prior to the removal of the plants from the culture medium.

PLANT REGENERATION PATHWAYS:

• Organogenesis: It is the process of morphogenesis involving the formation of plant organs i.e., shoots, roots, flowers, buds from explant or cultured plant tissues.
• Somatic embryogenesis: The process of regeneration of embryos from somatic cells, tissues or organs is regarded as somatic (or asexual) embryogenesis. Somatic embryogenesis may result in non-zygotic embryos or somatic embryos (directly formed from somatic organs), pathogenetic embryos (formed from unfertilized egg) and androgenic embryos (formed from male gametophyte).

SOMACLONAL VARIATION ('SPORTS')

It refers to genetic variations in plants that have been produced by plant tissue culture and can be detected as genetic or phenotypic traits.

Progeny that differ significantly from the parent are called 'somatic variants'. These somatic variants have established new and important varieties such as the naval orange or nectarines. Potato and sugarcane 'sports' were found to be resistant to disease in contrast to the susceptible parent plants. Herbicide resistance, cold- and salt- tolerance are additional favorable traits that can be found in 'sports'.

DEMENTIA

Dementia is defined as a global impairment or loss of intellectual function. It is a devastating disease that places a significant physical, emotional and financial burden on patients, their care taker and society.
It refers to a clinical syndrome characterized by progressive cognitive decline that interferes with the ability to function independently (particularly loss of short-term memory). Symptoms of dementia are gradual, persistent and progressive.
The clinical presentation of dementia varies greatly among individuals, and the cognitive deficits it causes can present as memory loss, communication and language impairment, agnosia (inability to recognize objects), apraxia (inability to previously learned task) and impaired execution function.
Dementia is not a specific disease. It is an overall term that describes a group of symptoms associated with a reduction in a person's ability to perform everyday activities.

Subtypes of Dementia:

Dementia is an overall term used to describe the clinical syndrome, but its subtypes are classified according to the cause of dementia. The four common types of dementia are:

• Alzheimer's disease
• Cerebrovascular disease
• Lewy bodies dementia
• Frontotemporal dementia

A. Alzheimer's disease

AD is the most common neurodegenerative disease responsible for dementia, comprising 60% to 80% of causes. It is believed to derive from the accumulation of beta-amyloid plaques and neuro-fibrillary tangles, first in the brain areas of the hippocampus which induces neuronal injury and subsequently, neuronal death. The resulting decrease in cholinergic transmission gives rise to loss of memory and cognition. More precisely neurotransmitters abnormalities include reduced activity of choline acetyl transferase and reduce the number of cholinergic neurons. Early onset AD is associated with autosomal dominant mutation in three genes: PSEN1, APP and PSEN2. Late onset AD is most commonly diagnosed in patients after the age of 60.

B. Cerebrovascular disease:

It is the second most prevalent form of dementia. Also called multi-infract dementia, it results from neuronal deprivation of oxygen caused by conditions that either block or reduce blood flow to the brain. Stroke is the most common cause. Its symptoms can vary widely, depending on the affected regions of the brain and the severity of the blood vessels damage. Some prominent symptoms include confusion, disorientation, vision loss, difficulty in speaking and understanding speech. Mixed dementia refers to the co-existence of AD and vascular dementia.

C. Lewy body dementia:

LBD is a form of dementia caused by abnormal deposits of alpha-synuclein protein (Lewy bodies) inside neurons. It accounts for 5% to 15% of all dementia. The most distinctive feature of LBD include fluctuation cognitive impairment with variations in attention and alertness, complex visual hallucinations and spontaneous Parkinsonism. Furthermore, rigidity and rapid eye movement (REM) sleep disorders are more commonly observed in early stages of LBD.

D. Frontotemporal dementia:

FTD is a general term used to describe disorders, such as Pick's disease, that affects the frontal and temporal lobes of the brain. FTD tends to occur at a younger age (40-75years) than does AD. Personality changes and behavioral disturbances are key features of FTD and occur early in the disease. In contrast to AD, visuospatial functions are usually not affected.

 CAUSES OF DEMENTIA:

Dementia can be caused by brain cell death and neurogenerative disease progressive brain cell death that happens overtime- is associated with most dementia.

But, as well as progressive brain cell death, like that seen in AD, dementia can be caused by head injury, a stroke or a brain tumor.

• Vascular dementia is resulting from brain cell death caused by conditions such as cerebrovascular disease. e.g., stroke. This prevents normal blood flow, depriving brain cells of oxygen.
•  Post traumatic dementia is directly related to brain cell death caused by injury. Some types of traumatic brain injury particularly, if repetitive. such as those received by athletes have been linked to certain dementias appearing late.
• Prion disease such as  CID (Creutzfeldt-Jakob Disease) is a fatal brain disorder which leads to dementia.
• HIV Viruses damage the brain cell.
• Deposits of amyloid, called plaques, build up around brain cell. Deposits of tau from 'tangles' within brain cell.
• There is also decrease in neurotransmitter like, acetylcholine in the brains of people of dementia.
• The hippocampus is often affected early in AD.
• People who smoke or have high blood pressure or diabetes have vascular dementia due to the narrowing of small blood vessels (subcortical vascular dementia).
• "Strokes" where part of the blood supply to brain is suddenly cut off is called post-stroke dementia. Lots of mini-strokes cause widespread damage to brain and is known as multi-infract dementia.
• Tiny clumps of a protein called alpha-synuclein than can develop into the brain cell and damage the cells work and communication, eventually dies. 
• Moreover, there are more than 50 identified causes of dementia including various infectious diseases; metabolic disorders (Wilson disease and Leukodystrophies); malignancies; epilepsies; traumatic brain disease; systemic disorders such as endocrine disease and vitamin deficiencies; toxic disorders including alcohol dependency and drug toxicity as well as mental illness such as depression and schizophrenia. 

RISK FACTORS FOR DEMENTIA:

Some dementia risk factors are difficult or impossible to change. These include:

AGE: 

Older people are more likely to develop dementia. However, dementia is not an evitable part of ageing.

GENES:

Certain genetic factors are involved with some more unusual forms of dementia, for the most part, dementia develops as a combination of genetic and environmental factors such as smoking and lack of regular exercise.

REVERSIBLE FACTORS:

Some dementias can be treated by reversing causes, including medication, interactions, depression, vitamin deficiencies and thyroid abnormalities.

MEDICATION:

• Antihypertensive:   Some studies say AD and other dementias may be caused by high BP, since it can cause blood vessel damage through constriction. The etiology of vascular dementia includes hypertension, and thus, lowering BP with antihypertensive may have a positive effect in prevention of dementia.
• Steroid Hormones: Estrogen may also help in the prevention of dementia but cannot help when it is already present and when cognitive function is already impaired. It increases cerebral blood flow and is an anti-inflammatory agent, enhancing activity at the neuronal synapses in the brain. It may also help to increase brain activation in regions that are affected by dementia which is mainly the hippocampus region.
• NSAIDS (Non-steroidal anti-inflammatory drugs) : It can decrease the risk of developing AD and Parkinson's disease. NSAIDS inhibit formation of some inflammatory and prevent the deteriorating effects.

MOLECULAR BASIS OF DEMENTIA:

Frontotemporal dementia (FTD) is a clinical syndrome with a heterogeneous molecular basis. Familial FTD has been linked to mutations in several genes, including those encoding the microtubule associated protein tau (MAPT), progranulin (GRN), valosin- containing protein (VCP) and charged multivescicular body protein 2B (CHMP2B). The associated neuropathy is characterized by selective degeneration of the frontal and temporal lobes (frontotemporal lobar degeneration, FTLD), usually with the presence of abnormal intracellular protein accumulations. FTD is often associated with an extrapyramidal movement disorder (Parkinsonism or corticobasal syndrome) or with motor neuron disease (MND). Major subgroups include those characterized by the physiological tau, TDP-43, intermediate filaments and a group with cellular inclusions composed of an unidentified  ubiquitinated protein.

FTLD-tau : Tau is a microtubule associated phosphoprotein (MAP) that is abundantly expressed in both the central and peripheral nervous system. The tau gene is located (MAPT) on chromosome 17q21 and has two major haplotypes. H1 and H2, which are defined by a set of single nucleotide polymorphisms and a 238 base pair deletion in intron 9.

In AD, abnormally hyper phosphorylated tau is the major component of the neurofibrillary lesions (neuropil-threads and dystrophic neurites). The abnormal accumulation of intracellular tau is characteristic of a number of other neurodegenerative disorders collectively known as 'tauopathies'. A  number of tauopathies may be associated with clinical FTD (FTLD-tau), including Pick disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD) and hereditary frontotemporal dementia and parkinsonism linked to chromosome 17, as a result of MAPT mutations (FTDP-17T).

FTLD-TDP: TDP-43 is encoded by TARDBP gene on chromosome 1. It is involved in cellular processes such as microRNA biogenesis, apoptosis, cell-division, mRNA stabilization and regulation of neuronal plasticity of acting as neuronal activity response factor. The exon skipping and splicing inhibitory activity requires the C-terminal region of TDP-43, which interact with the hnRNP family. TAR DNA-binding protein 43 (TDP-43) was identified as the pathological protein in most common subtypes of FTD and amyotrophic lateral sclerosis (ALS). The dramatic change in subcellular distribution of TDP-43 from the nucleus to the cytoplasm in inclusion bearing cells suggests that loss of pivotal nuclear TDP-43 have a pathogenic role. Cortical inclusions in FTLD-TDP are selectively enriched for hyper-phosphorylated C-terminal fragments (CTFs). Overexpression of this CTF is the key feature of the formation of ubiquitinated and phosphorylated cytoplasmic aggregates.

SIGNS AND SYMPTOMS:

The symptoms of dementia vary across types and stages of the diagnosis. The most common affected areas include memory, visual-spatial, language, attention and problem-solving. Most types of dementia are slow and progressive. By the time the person shows signs of the disorder, the process in the brain has been happening for a long time. It is possible for a patient to have two types of dementia at the same time. About 10% of people have mixed dementia, which is usually a combination of AD and another type of dementia.

Neuropsychiatric symptoms that may be present are termed behavioral and physiological symptoms of dementia (BPSD) and these include:

• Balance problem
• Speech and language difficulty
• Trouble eating and swallowing
• Memory distortions
• Restlessness
• Perception and visual problems
• Agitation
• Depression
• Anxiety and Irritability
• Delusions or hallucination
• Changes in sleep or appetite
• Abnormal motor behavior.

When people with dementia are put in circumstances beyond their abilities, there may be a sudden change to crying or anger (catastrophic reaction). Psychosis and agitation also often accompany dementia.

Mild Cognitive Impairment:

The earliest stage of dementia is called mild cognitive impairment (MCI). 70% off those diagnosed with MCI progress to dementia at some point. MCI shows the symptoms of dementia late.

Alzheimer's Disease:

• Difficulties in forming new memories
• Regularly forgetting recent events
• Becoming increasingly repetitive
• Misplacing items and getting confused
• Loss of interest in daily activities, becoming upset 

Vascular Dementia:

• Problems in attention, planning and reasoning
• Personality changes- depression, apathy, emotional
• Movements problems
• Bladder problems- frequent urge o urinate
• People need help in eating, dressing or using toilets.

Lewy bodies Dementia:

• Muscle stiffness and tremors.
• Repeatedly, realistic and well-formed hallucinations
• Shouting out while sleeping
• Fainting and unsteadiness.

Frontotemporal Dementia:

• Lack of personnel and social awareness
• Over-eating
• Develop unusual beliefs, interests and obsessions
• Twitching of muscles

At early stages of dementia, the symptoms interfere with person's daily activities. The person usually score 20-22 on the mini-mental state examination (MMSE).

At middle stage, the symptoms experienced get worsen. Moderate dementia patient score 6-17 on the MMSE. Person requires assistance for personal care. 

At last stage, person requires 24 hours supervision to ensure safety. They does not recognize common danger and are unable to control their bladder or bowels. Does not recognize familiar faces or people.

DIAGNOSIS:

Clinicians can diagnose the syndrome of dementia based on history, examination and appropriate objective assessments, using standard criteria such as DSH-5. Dementia is typically diagnosed when acquired cognitive impairment has become severe enough to compromise social and occupational functioning.

Diagnosis criteria for dementia:

The presence of an acquired impairment in memory, associated with impairment in one or more cognitive domains including:

• Executive function (e.g., obstruct thinking, reasoning, judgement)
• Language (expressive or receptive)
• Praxis (learned motor sequence)
• Agnosies (ability to recognize objects, faces or info.)

Diagnosis may be aided by brain scanning technique i.e., brain biopsy (rarely recommended).

COGNITIVE TESTING:

There are some brief tests (5-15 min.) that have reasonable reability to screen for dementia. presently mini-mental state examination (MMSE) is the best studied and most commonly used with high sensitivity and specificity for separating moderate dementia from normal cognition. A patient with mild dementia usually have a score of 18-26 out of 30, those with moderate dementia scores 10-18 and severe scores less than 10.

Other tests include Abbreviated Mental Test Score (AMTs), Modified mini mental state exam (3MS) Cognitive Abilities Screening Instrument (CASI), the Trait Making test and the clock-drawing test.

LABORATORY TESTS:

Routine blood tests are usually performed to rule out treatable causes. These tests include vitamin B12, folic acid, TSH, C-reactive protein, full blood count, electrolyte, calcium, renal function and liver enzymes. Abnormalities may suggest vitamin deficiency, infection or other problems that commonly cause confusion or disorientation in elders.

IMAGING:

A CT scan or magnetic resonance imaging (MRI scan) is commonly performed. These can yield relevant info to other type of dementia such as stroke.

The functional neuroimaging of SPECT and PET are more useful in assessing long-standing cognitive dysfunction. The ability of SPECT to differentiate the vascular cause from AD, appear superior to differentiation.

CSF ANALYSIS:

Cerebrospinal fluid analysis in dementia is generally intended to exclude an infective or demyelinated disorder. CSF concentration is generally raises only when there is extensive neuronal loss occurring over a short time period.

• A comprehensive examination of blood hemostasis factors including platelet count, specific clotting factors and anti-phospholipid-antibodies may be helpful.
• In individuals with history of alcohol abuse, thiamine deficiency should be considered but is usually diagnosed on the basis of therapeutic response to an intravenous bolus of thiamine.

TREATMENT:

• MEDICATION

The following are used to temporarily improve dementia symptoms:

CHOLINESTERASE INHIBITORS: These medications including donepezil, rivastigmine (Exelon) and galantamine (Raza dyne) work by boosting levels of a chemical messenger involved in memory and judgement. Side effects can include nausea, vomiting and diarrhea . With these types of drugs deterioration of the disease could be delayed by at least 12 months.

MEMANTINE (Namenda) works by regulating the activity of glutamate, another chemical messenger involved in brain functions such as learning and memory. Sometimes, it is prescribed along with donepezil in combination drug for moderate to severe dementia.

PSYCHOTROPHIC DRUGS can be used as supportive therapy in the treatment of behavioral problems in dementia.

ANTI-ANXIETY MEDICATIONS OR ANXIOLYTICS such as lorazepam (Ativan) can ease anxiety or restlessness.

ANTIDEPRESSANTS like serotonin reuptake inhibitors (SSRIs), can improve low mood and irritability.

ANTIPSYCHOTIC MEDICINES such as ariprazole, olanzapine etc. can help control feelings and behaviors such as delusions, agitation.

Treatment of dementia begins with the treatment of the underlying disease. The underlying causes of nutritional, hormonal,  tumor-caused or drug related dementia may be  eversible to some extent.

THERAPIES:

• Reminiscence therapy
• Cognitive stimulation therapy (CST)
• Reality Orientation therapy 
• Behavior orientated therapy
• Emotion-oriented therapy
• Stimulation oriented therapy

LIFESTYLE CHANGES:

• Stay active and organized
• Focus on food.
• Prioritize good sleep
• Challenge the brain

  

Write the mode of action of following mutagens:

5-BU:

The pyrimidine 5-bromo-uracil is a thymine analog, the bromine at the 5- position being similar in several aspects to the methyl group at the 5-position in thymine. The presence of the bromine, however, changes the charge distribution and increases the probability of tautomeric shifts. In its more stable keto form, 5-BU pairs with adenine. After a tautomeric shift to its enol form, 5-BU pairs with guanine. After one round of replication, an AT-GC transition occurs. The presence of 5-BU also increases the sensitivity of the DNA to UV which itself is mutagenic.

EMS:

Compounds that transfer alkyl groups (CH3-, CH3CH2) to the amino or keto groups of the nucleotides are called alkylating agents. For e.g., Ethyl methane sulfonate (EMS) is a chemical mutagen, which alkylates the keto-group in the 6-position of guanine and forms the abnormal base O6-ethylguanine. During replication, DNA Pol. that catalyzes the process, frequently place thymine instead of cytosine, opposite O6-ethylguanine. Following subsequent rounds of replication, the original base pair G:C can become an A:T pair (a transition mutation).

Acridine Orange:

Intercalating agents such as acridine orange have dimensions that allow them to wedge between the stacked base pairs in DNA. In so doing, they increase the rigidity and alter the conformation of the double helix, possibly causing slight "kinks" in the molecules or they can distort a DNA strands to unwind. These changes in DNA structure can affect many functions involving replication, transcription and repair. In many cases, when DNA molecules containing intercalated acridines replicate, additions and deletions of from one to a few base pair occurs. These small additions and deletions, usually of a single base pair result in reading frameshifts for the portion of the gene distal to the mutation.

UV:

UV radiations are readily absorbed by certain substances such as purines and pyrimidines, which then enter a more reactive or excited state. The maximum absorption of UV by DNA is at a wavelength of 254 nm. Because of their lower energy, they penetrate tissues only slightly, usually only the surface of multicellular organisms. However, it is a potent mutagen for unicellular. The two major product of UV absorption by pyrimidines appear to be pyrimidine hydrates or dimers. Thymine dimers appear to cause mutations  indirectly in two ways: (a) Dimers apparently perturb the DNA double helix, distort the DNA conformation and interfere with accurate DNA replication, (b) Occasional errors are made during the cell processes for the repair of damaged DNA, such as DNA containing thymine dimers.

MUTATIONS:

What are mutations?

Sudden, heritable changes in the genetic material i.e., an alteration of DNA sequence, any base pair change in any part of a DNA molecule is mutation. It may comprise a single base pair substitution, deletion, insertion or major alteration in the structure of chromosomes. It may occur within a protein coding regions of a gene or within the introns and regulatory sequences. It may occur within germ line cells or in somatic cells. Mutations is the ultimate source of all genetic variation; it provides the raw material for evolution.

Missense mutation: 

A change in one nucleotide of a triplet within a protein coding region of a gene may result in the creation of a new triplet that codes for a different amino acid in the protein product. If this occurs, mutation is known as Missense mutation. It is a non-synonymous substitution and a point mutation. For e.g., sickle cell disease is caused by a single point mutation (a missense mutation) in the beta-hemoglobin gene that converts a GAG codon into GUG which encodes the amino acid valine rather than glutamic acid.

Non-sense mutation:

Mutation in which a triplet or sense codon that corresponds to one of the twenty amino acids specified by the genetic code is changed into a stop codon resulting in the termination of translation of the protein, this is known as nonsense mutation. It usually results out in a non-functional protein product. Some genetic disorders such as thalassemia and DMD result from nonsense mutation.

Frameshift mutation:

A mutation caused by the addition and deletion of a base pair in the DNA of a gene resulting in the translation of the genetic code in an unnatural reading frame. It cause subsequent codons to be changed these are called frameshift mutation because the frame of triplet leading during translation is altered. For e.g., 1) Crohn's disease- insertion of cytosine at position 3020, leads to a premature stop codon, shortening the protein that is supposed to be transcribed. 2) Tay-Sachs disease 3) Cystic fibrosis caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. There are two frameshift mutations in exon 7, one caused by a two-nucleotide insertion, CF1154insTC and the other caused by one-nucleotide deletion CF1213delT. These shifts are predicted to introduce UAA termination codons at residues 369 and 368.

Transition:

A transition is a point mutation that changes a purine nucleotide to another purine (A-G) or a pyrimidine to another pyrimidine (C-T). Transitions can be caused by oxidative deamination and tautomerization. Approximately, two out of three single nucleotide polymorphism (SNPs) are transitions.

Transversion:

Base pair substitutions involving the substitution of a purine for a pyrimidine and vice versa are called transversions. It can only be reversed by a spontaneous reversion. It is less common than transition. This type of mutation is more likely to cause amino acid sequence changes. Transversions are caused by ionizing radiations, strong chemicals.

Photoreactivation:

It is one of the three different mechanisms for the repair of DNA containing thymine dimers. in E.coli. Photoreactivation involves an enzyme that splits thymine dimers directly without the removal of any nucleotide. This enzyme will bind to thymine dimers in DNA in the dark, but it cannot catalyze cleavage of the bonds joining the thymine molecules without energy derived from visible light. The enzyme is also active on cytosine dimers and cytosine-thymine dimers. Thus, when ultraviolet light is used as an experimental mutagen, the treatment is usually carried out in the dark to maximize the mutation frequency.

Key features of genetic code.

Genetic code is a three letters (triplets) code defining the transfer of the information from nucleic acid to proteins. Codon is a successive string of three nucleotides. Nucleotides found in human DNA are adenine (A), thymine (T), guanine (G), and cytosine (C). In RNA, thymine is replaced by uracil (U).

Genetic code has 6 main features :

1. The genetic code is degenerate : There are 20 main amino acids but they can be coded for by 64 different triplet combinations of codons. It logically means that one amino acid is coded by more than one codon. We can say that the nucleotide at last position is the least important. Some important amino acids are coded just by one codon (methionine and tryptophan). On other hand, serine is coded by 6 possible combinations.
2. The genetic code is unambiguous: This feature is related to the first point one amino acid can be coded by several different codon; however each codon only codes for one amino acid not more. Hence, the unambiguity of genetic code.
3. The genetic code is almost universal :  The genetic code is pretty similar in most of the organisms. It means that codon which codes for methionine in human does the same in prokaryotes. This point is not exactly true as recently, scientists has discovered many expectations from this rule.
4. The codon is read in mRNA in a contiguous fashion. There are no punctuations.
5. AUG has dual functions. It codes for methionine and act as start codon.
6. The codon is triplet. 61 codons code for amino acids and three codons do not code for any amino acid. Hence, they function as stop codons. 

Describe the positive and negative control of lac operon.

The lac operon is responsible for the metabolism of lactose and is also induced by lactose.

Negative control of lac operon:

The lac I gene of lac operon encodes a repressor which, in the absence of lactose binds to the operator (o) and blocks the transcription of the three structural genes; 

Z ; beta-galactosidase

y; lactose permease

a; thiogalactosidase transacetylase

Lactose induces expression of the operon by binding to the repressor, which prevents the repressor from binding to the operator. The lac operator overlaps the promoter and so the repressor bound to the operator physically prevents RNA Pol from binding to the promoter. (Repressor-operator interactions).

Allolactose then acts as an inducer and binds to the lac repressor, leading to its dissociation from DNA and hence, the production of lac mRNA (and the 3 enzymes).

Positive control of the lac operon:

It is also called the catabolite control of lac operon. The initiation of lac mRNA synthesis is also regulated by the concentration of glucose. No lac mRNA is made in the presence of glucose.

Cyclic AMP (cAMP) is synthesized by the enzyme adenylate cyclase and its concentration is regulated indirectly by glucose. The entry of glucose in the cell makes the intracellular concentration of cAMP quite low. Under starvation conditions, the cAMP concentration in bacterial cells is high. It is cAMP that links the lac operon the activity with the intracellular concentration of glucose.

E.coli contains a protein called CRP (cAMP receptor protein). CRP and cAMP bind together to form a complex (denoted cAMP-CRP) which is the regulatory element in the lac system. The cAMP-CRP complex binds to a specific sequence called an activation site in the promoter region for transcription of the lac-operon to occur. Thus, the cAMP-CRP complex is a positive regulator (activator) of the lac operon.

Attenuation of trp operon

Bacteria such as E.coli need amino acid to survive because, like us, they need to build protein. One of the amino acid they need is tryptophan. If tryptophan is available in the environment, E.coli will take it up and use it to build protein. However, E.coli can also make their own tryptophan using enzymes that are encoded by five genes. These five genes are located next to each other in what is called the trp operon.
Like regulation by trp repressors, attenuation is a process for reducing the expression of trp operon when levels of tryptophan is high. However, rather than blocking initiation of transcription, attenuation prevents completion of transcription.
When levels of tryptophan are high, attenuation causes RNA Polymerase to stop prematurely when it is transcribing the trp operon. Only a short, stubby mRNA is made, one that does not encode any tryptophan biosynthesis enzymes. Attenuation work through a mechanism that depends on coupling.
The structure of trp operon consists of operator and first gene of the operon is called leader. The leader encodes a shorter polypeptide and also contain an attenuator sequence . The attenuator does not encode a polypeptide, but when transcribed into mRNA, it has self-complementary sequences that can form various hairpin structures.
Once RNA Pol has started transcribing the operon, a ribosome can attack to the still-forming transcript and begin translating the leader region. The polypeptide encoded by leader is short, just 14 amino acids long and it included two tryptophan residues.

• If there is plenty of tryptophan, the ribosome won't have to wait long for a tryptophan carrying tRNA, and will rapidly finish the leader polypeptide.
• If there is little tryptophan, the ribosome will stall to at trp codons (waiting for a tryptophan carrying tRNA) and will be slow to finish the translation of the leader.

As we see earlier, the leader is followed by an attenuator region, which (in its mRNA form) can stick to itself to form  different hairpin structures. One structure include a transcription terminal signal, while the other does not end termination.

• If the ribosome translates slowly, it will pause and its pausing causes formation of anti-terminator (non-terminating hairpin). This hairpin prevent formation of terminator and allow transcription to continue.
• If the ribosome translate quickly, it will fall off the mRNA after translating the leader peptide. This allow the terminator hairpin and associated hairpin to form, makin RNA Pol detached and ending transcription. 

This mechanism may be complex, but the result is pretty straightforward. When tryptophan is abundant, the ribosome moves quickly along the leader, the terminator hairpin form and transcription of trp operon end. When tryptophan is scare, the ribosome moves slowly along the leader, the non-terminator hairpin form and transcription of the trp operon continues.

In other words, the logic of attenuation is the same as that of regulation by the trp repressor. In both cases, high level of tryptophan in cell shut down the expression of operon. This makes sense, since high level of tryptophan means cell does not need to make more biosynthetic enzymes to produce additional tryptophan.

Differences between inducible and repressible operon:

INDUCIBLE OPERON:

1. In inducible operon, the genes are kept switched off until a specific metabolite inactivates the repressor.
2. It operates in catabolic pathway.
3. It starts transcription and translation.
4. It is caused by a new metabolite, which needs enzymes to get metabolized. Thus, the nutrients utilized in the pathway activate enzyme synthesis.
5. Repressor is prevented by the inducer from joining the operator gene.
6. E.g., lac operon is an inducible operon.
7. Without lactose, E.coli do not produce beta-galactosidase, lactose permease, and transacetylase. When lactose is added to the medium, E.coli start producing the enzymes.

REPRESSIBLE OPERON:

1. In repressible operon, genes are kept switched on until the repressor is activated by a specific metabolite.
2. It operates in an anabolic pathway.
3. It stops transcription and translation.
4. The production is switched off by the end products of the pathway which repress enzyme synthesis. It is caused by an excess of existing molecules.
5. Apo-repressor is enabled by a co-repressor to join the operator gene.
6. E.g., trp operon is an repressible operon.
7. Without tryptophan, E.coli produce a lot of tryptophan. when tryptophan is added to the medium, E.coli stop producing tryptophan. 

Differences between LFT and HFT.

LFT:

1. Low frequency transducing lysate.
2. A lysate formed from a single lysogeny in which aberrant excision occurs only infrequently is called a LFT lysate. Small number of specialized transducing phages are produced and mostly contain normal phages.
3. Single lysogeny produces transducing particles at about 10^(-6) to 10^(-7) of the total phage particles in the lysate.
4. No involvement of helper phage.
5. 99.9% non-transducing particles of the total particles.

HFT:

1. High frequency transducing lysate.
2. Lysates containing a relatively large number of specialized transducing phages created by coinfecting a host cell with a helper (normal) phage and a transducing phage- dilysogen.
3. The dilysogens yield lysates half of whose phage are transducing particles.
4. Helper phage allows the transducing phage to replicate, thus increasing the number of transducing phages in the lysate.
5. 50% non-transducing out of the total particles.

Differnces between generalized and specialized transduction

GENERALIZED TRANSDUCTION:

1. Any piece of the bacterial genome can be transferred.
2. It is done by virulent or lytic bacteriophages.
3. Undergoes lytic cycle.
4. Bacterial cell lyses quickly.
5. Generalized transducing phage produce some particles that contain only DNA obtained from the host bacterium, rather than phage DNA.
6. Bacterial DNA hydrolyses by the virus into pieces.
7. E.g., E.coli by phage 1.
8. No-prophage formation.

SPECIALIZED TRANSDUCTION;

1. Only specific pieces of the chromosome can be transferred.
2. It is done by temperate phage.
3. Undergoes lysogenic cycle.
4. Cell survive for several generations.
5. A specialized transducing phage produces particles containing both phage and bacterial genes linked in a single DNA molecule.
6. Bacterial DNA is not hydrolyzed.
7. E.g., lambda-phage.
8. Prophages are formed.

Differences between F and HFr plasmid

F-Plasmid:

1. F-plasmid is fertility factor, it contains the genes require for the transfer or conjugation.
2. F(+) * F(-) ……… recipient becomes F(+).
3. Low rate of recombination .
4. It takes 2 minutes for the transfer of F-plasmid.
5. One strand of the F factor is nicked and moves across the conjugation tube. The DNA complement is synthesized on both strands. The entire F-plasmid factor moves in the recipient cell.
6. The transferred F-factor synthesizes its DNA complement and get circularize independently in the recipient cell. 

HFr-Plasmid:

1. HFr-plasmid is a special case of F(+) cells. i.e., a cell with an integrated F.
2. HFr * F(-) ……… recipient becomes F(-).
3. High rate of recombination .
4. It takes 100 minutes for an entire chromosome to be transferred.
5. During transfer of Hfr DNA to a recipient cell, the mating pair usually breaks apart before the entire chromosome is transferred.
6. In Hfr transfer, the transferred DNA fragment does not circularize and cannot replicate but one or more of its regions is frequently exchanged with the chromosome of the recipient, thereby generating recombinants in the F(-) cells.

Mechanisms of genetic exchange

1. Competence:

It is the ability of a cell to take up extracellular (naked) DNA from its environment. It results from changes in cell wall of bacteria. It usually arises at the late log phase. On the basis of the conditions under which the bacteria uptakes the DNA, it can be differentiated into natural competence and induced/ artificial competence.

  2. Lytic phage:

The phage which causes lysis of the host is called a lytic or virulent phage. An example of a lytic bacteriophage is T4, which infects E.coli found in the human intestinal tract.

3. Temperate phage:

Temperate phage are basically bacteriophages that can choose between a lytic and lysogenic pathway. They can either lyse the cell or behave as a prophage (viral DNA integrated into the bacterial chromosome).

4. F' plasmid:

F plasmid is an episome so once it integrated  into host chromosomal DNA, it can also disintegrate itself back from the host chromosome. F factor frequently carries several adjacent bacterial genes next to its disintegration sites along with it. This condition is called F' plasmid. They are the derivatives of Hfr plasmid.

5. Merozygote:

When a bacterium with F' conjugates with F- (-ve) cells, the F factor containing the chromosomal genes, is transferred to the F- cells. Thus, the chromosomal genes that are part of the F factor are now present as duplicate in the recipient cell. This creates a partially diploid cell called a merozygote.

6. Prototroph:

A bacterium must be able to synthesize all essential organic compounds. A bacterium that can accomplish this remarkable biosynthetic feat- one that the human body cannot duplicate- is a prototroph. It is said to be wild type for all growth requirements.

7. Auxotroph:

If a bacterium loses, through mutations the ability to synthesize one or more organic compounds, it is an auxotroph. It is mutant organism that requires a particular additional nutrient which the normal strain does not.

8. Abortive transduction:

It is an event of transduction in which the transducing DNA (genetic fragment from the donor bacterium) fails to be incorporated into the recipient chromosome, and when the latter divides, is transmitted to only one of the daughter cells.

9. Co-transduction frequency:

The co-transduction frequency is the ratio of transductants that co-inherit both markers divided by the total number of transductants. Basically, only a small amount of chromosome, a few genes, can be transferred by transduction. The closer two genes are to each other, the more likely they are to be transduced by the same phage. Thus, "co-transduction frequency" is the key parameter used in mapping genes by transduction.

The Wu formula can be used to estimate the correlation between co-transduction frequency and the physical distance between two genetic markers.

          co-transduction frequency= (1-d/L)^3

where, d= distance between two genes in minutes

L= the size of the transduced DNA (in minutes)

 

Differnces between organization of prokaryotic and eukaryotic DNA:

Organization of Prokaryotic DNA:

1. Found in the nucleoid region.
2. Prokaryotes generally have a small, circular DNA.
3. There is a single origin of replication.
4. Less elaborately structured and folded genome.
5. The genome size ranges in between 10^4 to 10^7 bp with a high gene density.
6. They are polycistronic i.e., multiple genes are transcribed through the same promoter.
7. A single RNA Polymerase is involved in transcription.
8. Multiple proteins act together to fold and condense prokaryotic DNA. Folded DNA is then organized into a variety of conformations that are supercoiled and wound around tetramers of the HU proteins.

Organization of Eukaryotic DNA:

1. Found in the nucleus.
2. They have multiple linear chromosomes (2 to <50.
3. The chromosomes have multiple origin of replication sites.
4. Complexed genome with a large amount of protein to form chromatin.
5. The genome size ranges from10^8 to 10^11 bp with a low gene density due to the presence of introns.
6. They are monocistronic i.e., one gene is under the control of one promoter.
7. Three type of  RNA Polymerase are present of which RNA Pol II is involved in transcription.
8. Eukaryotes wraps their DNA around proteins called histones .

Differentiate between denaturation and renaturation of DNA.

DENATURATION OF DNA:

1. The process of formation of single stranded DNA from double stranded helical DNA.
2. It is effected by heating.
3. It involves breakage of hydrogen bonds between complementary base pairs.
4. The increase in absorbance (A260) upon denaturation is called as hyperchromic effect.
5. The rate of increase in absorbance is directly proportional to the rate of denaturation.
6. Upon denaturation, viscosity decreases.

RENATURATION OF DNA:

1. The process of formation of double stranded DNA from single stranded complementary DNA strands.
2. It is effected by cooling.
3. It involves reannealing or formation of hydrogen bonds between complementary base pairs.
4. There is decrease in absorbance (A260) upon renaturation .
5. The rate of renaturation is directly proportional to the concentration of complementary sequences.
6. Upon denaturation, viscosity increases.

What are the postive and negative effects of transposons?

NEGATIVE EFFECTS:

There are multiple negative effects of transposons on phenotypes:

• If transposon insert themselves into a coding region or into a regulatory sequence, they will most probably render that gene inactive.
• Upon leaving a site, the gap that is left by a transposon will need to be repaired, this repair, often, will cause genetic mutations in the original gene and render it inactive.
• Sequences that are repeated multiple time leads to unequal crossing over and chromosomal abnormalities.
• A number of diseases in the genome have been linked to transposons, such as Hemophilia A and B Porphyria. Severe Combined Immunodeficiency (SCID), predisposition to cancer and Duchenne Muscular Dystrophy.

Despite these negative aspects there can be positive impacts of transposable elements:

POSITIVE EFFECTS:

• Sometimes non-transposons, coding, DNA gets carried along with mobile elements during transposition; this would lead to the duplication of beneficial genes or the creation of new genes.
• Sometimes genetic mutation caused by transposons may modify regulatory sequences and this could change the pattern of expression of gene produced, which could lead to some new beneficial characteristics.
• The rate of transposition has actually been observed to increase in some cases under the conditions of stress. This higher rate of transposition could cause genetic mutations leading to new traits, which would allow an organism to adapt better to changing environmental conditions.
• Transposons have also come in handy as tools for scientists who wish to create transgenic organisms or modify an organism DNA for research purposes.

USES OR APPLICATIONS OF PLASMID:

IN CLONING:

Plasmids are the most commonly used bacterial cloning vectors. These cloning vectors contain a site that allows DNA fragments to be inserted. After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called transformation. These plasmids contain a selectable marker, usually an antibiotic resistance gene, which confer on the bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics. The cell after transformation are exposed to the selective media, and only cells containing the plasmid may survive. The vector may also contain other marker genes to facilitate selection of plasmid with cloned vector insert. Bacteria containing the plasmid can then be grown in large amounts, harvested and the plasmid of interest may then be isolated. A plasmid cloning vector is typically used to clone DNA fragments of up to 15 kbps. These cloning vectors can be plasmids, cosmids, bacterial artificial chromosome (BACs), yeast artificial chromosomes (YACs), or bacteriophages (phage-lambda and M13 phage) e.g., of two known cloning vectors are pBR322 and pUC19.

EXPRESSION:

Another major use of plasmids is to make large amount of proteins. Expression vector (plasmids) are used to introduce a specific gene into a target cell and commander cell's mechanisms produce a relevant gene product. It comprises of enhancers, promoter region, termination codon, transcription initiation sequences and translation initiation sequences in addition to features of a cloning vector. In this case, as the bacteria produces proteins to confer its antibiotics resistance, it can be induced to produce large amounts of proteins from the inserted gene. For e.g., insulin Example of expression vector is pET28a.

MUTAGENESIS:

It is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of gene and any gene product. It is also called site specific mutagenesis or oligonucleotide directed mutagenesis. It is used for investigating the structure and biological activity of DNA, RNA and proteins.

Explain the vector map of:

pBR322

pBR322 is a plasmid and one of the first widely used E.coli cloning vectors. It was named after the researcher who constructed it "Bolivar" and "Rodriguez".

• It is 4361 base pairs in length.
• It has two antibiotic resistance genes- the gene bla encoding the ampicillin resistance (AmpR) protein, and the gene tetA encoding the tetracycline resistance (TetR) protein.
• It contains the origin of replication of pMB1, and the rop gene, which encodes a restrictor of plasmid copy number.
• It has unique restriction sites for more than forty restriction enzymes (11 out of 40 sites lie within TetR gene), (2 sites for restriction enzyme- Hind III and Cla I are at promoter of TetR gene). There are 6 restriction sites inside the AmpR gene.
• The circular sequence is numbered such that O is the middle of the unique EcoRI site and the count increases through the TetR gene.
• The AmpR gene is penicillin beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene.
• P3 is the natural promoter and P1 is artificially created by the ligation of two different DNA fragments to create pBR322.
• P2 is in the same region as P1, but it is on the opposite strand and initiates transcription in the direction of tetracycline resistance gene.
• Copy number- 1-100 per cell.

pUC:

pUC is one of the series of plasmid cloning vectors. "p" prefix denoting plasmid and the abbreviation UC for the University of California, where early work on the plasmid series had been conducted.

• It is a circular double stranded DNA and has 2686 base pairs.
• It has one ampR gene (ampicillin  resistance gene) and an N-terminal fragment of beta-galactosidase (lacZ) gene of E.coli.
• The multiple cloning site (MCS) region is split into the lacZ gene (codons 6-7 of lacZ are replace by MCS), where various restriction sites for many restriction endonuclease are present.
• In addition to beta-galactosidase, pUC19 also encodes for an enzyme called beta-lactamase, which can degrade ampicillin and reduces its toxicity to the host.
• The ori site or origin of replication is derived from the plasmid pMB1.
• pUC19 is small but has a high copy number. The high copy number is a result of the lack of the rop gene and a single point mutation in the ori of pMB1.
• The recognition sites for HindIII, SphI, Pst I, Sa II, Xba, BamtII, SmaI, KpnI, SacI and EcoRI restriction enzyme have been derived from the vector M13mp19.
• When the pUC plasmid has been used to transform the host cell E.coli the gene may be switched on by adding the inducer IPTG. Its presence causes the enzyme beta-galactosidase to be produced which will hydrolyze a colorless substance X-gal into a blue soluble material. However if the gene is disrupted by the insertion of a foreign fragment of DNA, a non-functional enzyme results which is unable to carry out the hydrolysis of X-gal. Hence, recombinant can be easily distinguished from the non-recombinants based on the color differences of colonies on growth media.
• pUC vectors carry different combinations of restriction sites and shows greater flexibility in the types of DNA fragment that can be cloned. Clustering of the restriction sites allow a DNA fragment with two different sticky ends to be cloned without involving linker attachment.
• 

pET28a

• Size- 7328 base pairs.
• It has bacterial expression vector with T7 lac promoter, adds N-terminal His tag, thrombin cleavage site, internal T7 epitope tag, C-terminal His tag, restriction enzyme cloning.
• It has antibiotic resistance for kanamycin.
•  pET28a is used for bacterial expression, in vitro transcription, multiple cloning site, with tag/fusion/marker.

 

Pathophysiology of Vitamin D

There are three main diseases caused by the deficiency of vitamin-D which are given as follows:

RICKETS:

Rickets is a disorder caused by the deficiency of vitamin D, calcium or phosphate. Rickets lead to softening or weakening of the bones and is seen most commonly in children 6-24 months of age. There are several subtypes of rickets, including hypophosphatemia rickets (vitamin-D-resistant rickets), renal or kidney rickets (renal osteodystrophy), and most commonly, nutritional rickets (caused by dietary deficiency of vitamin D, calcium or phosphate). Classic nutritional rickets is also medically termed as osteomalacia.

CAUSES:

Regardless of the type of rickets, the cause is always either due to deficiency of vitamin D, calcium or phosphate. Three common causes of rickets include nutritional rickets, hypophosphatemic rickets, and renal rickets.

Nutritional rickets

Nutritional rickets, also called osteomalacia, is a condition caused by vitamin D deficiency. Infants and children most at risk for developing nutritional rickets include dark-skinned infants, exclusively breastfed infants, and infants who are born to mothers who are vitamin D deficient. In addition, older children who are kept out of direct sunlight or who have vegan diets may also be at risk.

Hypophosphatemia rickets

Hypophosphatemia rickets is caused by low levels of phosphate. The bones become painfully soft and pliable. This is caused by a genetic dominant X-linked defect in the ability for the kidneys to control the amount of phosphate excreted in the urine. The individual affected is able to absorb phosphate, calcium but the phosphate is lost in the urine.

Renal (kidney) rickets

Similar to hypophosphatemia rickets, renal rickets is caused by the number of kidney disorders. Individuals suffering from kidney diseases often have decreased ability to regulate the amount of electrolytes lost in the urine. This includes calcium and phosphate, and therefore, the affected individuals develop symptoms almost identical to severe nutritional rickets.

SYMPTOMS:

Signs and symptoms of rickets include bone pain or tenderness, dental deformities, delayed formation of teeth, decreased muscle strength, impaired growth, short stature, and a number of skeletal deformities, including abnormally shaped skull (craniotabes) bowlegs, rib-cage abnormalities (rachitic rosary) and breastbone pelvic and spinal deformities.

DIAGNOSIS:

Rickets is initially diagnosed clinically with a complete medical and nutritional history and with a complete physical exam by a health professional. If rickets is suspected in a child and the child has no acute symptoms such as seizures or tetany, X-rays of long bones (radius, ulna and femur) and ribs are obtained.

TREATMENT:

The treatment for rickets depends upon the cause as mentioned above in the discussion of hypophosphatemia rickets and renal rickets. In cases of nutritional rickets and vitamin D deficiency, treatment is simple. Once the diagnosis of rickets is confirmed, initiation of vitamin D supplementation is recommended as well as a diet rich in calcium. This is especially important for children on vegan diets, The treatment for some of the bony abnormalities depends on the severity of the cases and may require referral to the orthopedic provider for evaluation.

OSTEOMALACIA

Osteomalacia is a weakening of the bones due to problems with bone formation or the bone building process. It is not the same as osteoporosis, which is weakening of living bone that has already been formed and is being remodeled.

CAUSES

The most common reason that osteomalacia occurs is a lack of vitamin D. A diet that doesn't include phosphates can result in phosphate depletion, which can also lead to osteomalacia.

Certain drugs, such as phenytoin and phenobarbital used to treat seizures, can also cause osteomalacia.

 SYMPTOMS

Bones that fracture easily are the most common symptom of osteomalacia.

Another symptom is muscle weakness due to problems at the location where the muscle attaches to bone. You may have hard time walking and may develop a waddling gait.

Bone pain, especially in the hips, is also common symptom. This dull, aching pain can spread from the hips to the lower back, pelvis, legs and even ribs. Low blood calcium may also cause numbness around the mouth or in your arms, legs and spasms in hands and feet.

DIAGNOSIS

Blood tests to check for low of vitamin D, calcium, and phosphates in blood can help diagnose osteomalacia and other bone disorders. May be tested for alkaline phosphatase isozymes. High levels of these indicate osteomalacia. Another blood test can check your levels of PTH, high levels of which are associated with insufficient vitamin D and other related problems.

X-rays and other imaging tests can reveal small cracks in the bones throughout the body. These cracks are called Looser's transformation zones. These cracks are where fractures can begin with even small injuries.

A bone biopsy may be required to definitively diagnose osteomalacia. A needle is inserted through the skin and muscle and into your bone to obtain a small sample. That sample is put on a slide and examined under a microscope. Usually, an X-ray and blood test are sufficient to make a diagnose and bone biopsy is not necessary. 

TREATMENT:

Treatment can be as simple as taking oral supplements for vitamin D, calcium or phosphate. If you have absorption problems due to intestinal injury or surgery, or have a diet low in key nutrients, this may be the first line of treatment. In rare cases, vitamin D may be administered as an injection through the skin, or intravenously through a vein in your arm.

OSTEOPOROSIS

It is a progressive bone disease that is characterized by a decrease in bone mass and density and that leads to an increased risk of fracture.

CAUSES:

Osteoporosis occurs when bone tissue and minerals are lost faster than the bone is replaced.

There are two main types of osteoporosis: primary and secondary.

Primary osteoporosis

It occurs most commonly in women after menopause. Osteoporosis affects twice as many females over the age of 70 years as males in the same age group.

Secondary osteoporosis

It can affect young and middle aged people as well. It may be caused by :

• medications such as corticosteroids (e.g., prednisone)
• chronic illness such as anorexia nervosa (eating disorder leads to malnutrition)
• too much exercise- women who exercise excessively lose their menstrual cycle and normal production of estrogen by the ovaries may stop.

SYMPTOMS:

Weakened bones that are no longer able to support body weight can break even under slight pressure. Such fractures most commonly occur in the hipbones, wrists or spine. Hip fractures are more frequent in people over the age of 75 years.

Some fractures caused by osteoporosis, such as hairline breaks in the spine, may cause little or no pain and may go unnoticed, even when they show up on an X-ray. By contrast, spinal crush fractures, where the vertebral column crumbles or collapses, are much more painful and can lead to deformed posture.

Another symptom caused by osteoporosis is chronic back pain. This pain can worsen even when you are making small movements such as regular activities around the house, or while coughing, laughing, or sneezing. You may even feel pain when you are standing still.

DIAGNOSIS:

Bone density measurement by a method called DEXA (dual energy X-ray absorptiometry) is the most effective way to assess osteoporosis risk.

A heel ultrasound test may be used to test bone density and estimate the risk of fracture for women over 65 years of age.

TREATMENTS:

Hormone replacement therapy (HRT) consisting of estrogen alone, estrogen and progesterone, has clearly been found to be useful in reducing menopausal vasomotor symptoms and in reducing the incidence of skeleton fractures. National Institute of Health (NIH) study suggested that HRT may be associated with increased risk of breast cancer in women with a uterus and increased rate of thromboembolic events and stroke.

Three year of 1,25 (OH)2 D3 therapy in woman with post menopausal osteoporosis significantly reduced the incidence of new vertebral fractures as compared with Ca2+ gluconate supplementation.