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Functional Assay of Cytokine

2020-06-04T12:47:00.001+05:30

Functional Assay of Cytokine

Function assay of cytokines is used to monitor to activity of different types of cytokines this cytokine can induce either growth or death in target cell. There are two type of function assay of cytokines namely growth assay and cytotoxic assay.

Growth assay: -

This type of assay is generally use to check the activity of IL-2. IL-2 is a mitogen which induce mitosis in the target cell the assay is perform as follows.

Protocol: -

- First a 96 well microtiter plate is taken and media required for growth of inducer cells is equally distributed in microtiter plate. Generally, RPMI media is used.

- After this the indicator cells are distributed or added in each well (100 micro lit of cell of 3×10⁵ cell/ml concentration)

- After cytokine sample or IL-2 sample is double diluted and each dilution is added to well.

- The microtiter plate is the kept for incubator 24 to 48 hours at 5% concentration of Co₂ in humified incubator.

- During incubation IL-2 induces death in the target cell according to its concentration and target activity.

- After incubation the plate is removed and MMT indicator dye is equally distributed in each well MMT dye is yellow color tetrazolium dye (2,3,5-tetraphenyl-2H-tetrazolium chloride) which is present in its oxidized from.

- The NADP dependent oxidoreductase enzyme present in the indicator cells reduces this dye to form purple colored produced known as formazan.

- This intensity of purple color is dependent on number of living cells in the sample.

- The intensity, concentration of purple color (ultimately the cell) can be determined by reading the plate at 560-570nm.

- The standard graph plot is generated and concentration can be determined.

Cytotoxic assay: -

This type of assay is generally used to check the activity of Interleukin-17, which has to be yet to be completely characterized, through membrane cytokines have a specific effect in promoting proliferation of T cell that cause cytotoxic cell. Interleukin-17 is a necrogen which induces necrosis in target cell (i.e. cell death)

Protocol: -

- First a 96 well microtiter plate is taken and media required for growth of inducer cells is equally distributed in microtiter plate. Generally, RPMI media is used.

- After this the indicator cells are distributed or added in each well (100 micro lit of cell of 3×10⁵ cell/ml concentration)

- After cytokine sample or IL-17 sample is double diluted and each dilution is added to well.

- The microtiter plate is the kept for incubator 24 to 48 hours at 5% concentration of Co₂ in humified incubator.

- During incubation IL-17 induces death in the target cell according to its concentration and target activity.

- The NADP dependent oxidoreductase enzyme present in the indicator cells reduces this dye to form purple colored produced known as formazan.

- This intensity of purple color is dependent on number of living cells in the sample.

- The intensity, concentration of purple color (ultimately the cell) can be determined by reading the plate at 560-570nm.

- The standard graph plot is generated and concentration can be determined.

Expression of Recombinant Protein Animal Cell Culture

2020-06-01T16:02:00.004+05:30

Expression of Recombinant Protein Animal Cell Culture

- Animal cell culture now days by using technology and the animal cell or tissue are grown in laboratory by using selective media.

- Animal cell culture is widely used for production of various pharmaceutical products of monoclonal antibodies.

- For the making of recombinant protein the gene is isolated and cloned into an expression vectors.

- Most of recombinant product are produced from bacteria yeast or animal cell in culture.

- Expression of recombinant protein in animal cell in culture is clone by inserting the gene of interest into the host cell

- Vector, which then donned into the host cell after this expansion of selected done is done.

Example

Mammalian cell	The ambolytic enzyme, monoclonal antibodies, hormones
Namalwa cell line	interferons

Advantages: -

1. It shows post translation modification.

2. High level of expression protein.

3. Many proteins produced naturally in now produced in animal cells.

Disadvantages: -

1. complex nutritional media require.

2. Growth rate is slow.

3. Susceptible to physical damage.

4. High production cost.

5. Recovery process is difficult.

Bacterial Toxins

2020-05-31T11:36:00.000+05:30

BACTERIAL TOXINS

Toxin are major determinants of virulence

There are two two type toxin such as

1. Endotoxins [secreted by Gram negative bacteria (Lipid A)]

2. Exotoxin

Endotoxins: -

This are the cell wall component of Gram-negative bacteria, which generally involved in pyrogen also in inflammation.

Exotoxin: -

This are the protein glycoprotein which are secreted by bacteria which are generally enzymes and alter host metabolism

e.g. Diphtheria toxins, cholera toxins, tetanus toxins.

Cholera toxin: -

- Produce by vibrio cholera a facultative anaerobe which caused disease cholera.

- Cholera toxin also known as cholerogen

- The virulence gene are clustered in chromosomal segment called as CTS box.

Gene	Product	Function
Ctx AB	Choler’s toxin	Main toxin in cholera
Zot	Cumulus acuminous	Active Polymerization
Aco	Accessory toxin	Hyper recreation

- Out of these three toxins, the gene product of ctx AB the main cholera toxin and other two toxin preferable accessory toxin

- The expression of this gene controlled by another gene tax, R.

- The cholera toxin and type 4 pili are key virulence factor from this bacterium

- Type 4 pili acts as adhesion molecule which bring the vibrio in close contact of mucous surface

Structure of cholera toxin: -

- Cholera toxin is typical AB type toxin in which there are 1 subunit of A and 5 subunits of AB

- The molecule weight of A is 23500 and B is 11500

- The Gm^-1 ganglioside present on crypt cell and villus enter site of intestine serve as receptor for cholera toxin

- The subunit A is made up of two A₁and A₂ which are connected each other by disulfide linkage

Mechanism of Action: -

- When cholera toxin is secreated bind to the Gm^-1 ganglioside and A subunit enters into cell.

- Once enter into the cel the disulphide linkage behaves A_g and A₂ is reduced and two subunit are released from each other

- The subunit A₁ is active cholera toxin

- The alter the normal function of G protein

- The G protein in are involved in signal transduction and ultimetly regulates activation and inactivation at an enzyme which regulates activation and inactivation of an enzyme which regulates the metabolism of cell [Adenylate Cyclase] which regulates the production of cyclic AMP

- Under normal condition the Amp is produced in this a signal molecule binds to receptor present on cell to release of GDP and binding of GTP

- The activated G protein (αG protein) then bind to adenylate cyclase and activate it and this activated adenylate cyclase synthesis of CAMP from ATP but when GTP is hydrolyze the αG protein is released form adenylate cyclase and adenylate cyclase is deactivation of this toxin (A₁) permanently activates adenylate cyclase enzyme by altering the activity of G protein

- Cholera toxin catalyze ADP ribosylation of αG protein (GTP bound form) which is bound to adenylate cyclase permanently.

- Therefore, concentration of CAMP increases in cell

- The CAMP regulate the ion traffic and its increased concentration, the cell has 3 effect on ion migration

1] Electrogenic chlorine pump are stimulated causing high amount of cl^- leaving the cell.

2] Na⁺, H⁺exchange system is blocked.

3] The increased cl^- in lumen intestine increases the HCO₃^-

- The ultimate result of this process is increased concentration of cl^-, HCO₃ and Na⁺in lumen of intestine [outside cell]

- Therefore, the water present in cell comes out in the lumen

- Therefore, affected patient excrete 1lit of fluid/day

- This result in loss of bodies fluid in feces

- The normal person excrete only 100ml fluid in faces 1 day

RFLP (Restriction Fragment Length Polymerization)

2020-05-28T22:15:00.005+05:30

RFLP (Restriction Fragment Length Polymerization)

Principle: -

Restriction fragment length polymerization (RFLP) technique in which organism may be differentiated by analysis of pattern derived from cleavages of this DNA

- If two organism differ in this distance between sites of cleavage of particular restriction endonucleases, the length of the fragment produced wise differ when the DNA is digested with a restriction enzyme.

- The similarity of the pattern generated can be used to differentiated species (and even strain) from one other.

Introduction: -

Alc Jeffery in 1985 discovered the technique called restriction fragment length polymerization (RFLP)

- RFLP is technique that exploits variation in homologous DNA sequences.

- It refers to a difference between sample of homologous DNAmolecule that come from differing location of restriction enzymes sites and to related laboratory technique locating of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated

- RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application

Procedure: -

It uses various produces like PCR, digestion of DNA with restriction enzyme endonuclease, Agarose gel electrophoresis, radiolabeling of DNA with probes, southern blotting, Autoradiography.

1] PCR: -

to analyze the DNAs with the keep of RFLP it requires large amount of DNA for a better detection. Therefore, the DNA to be analyzed can be amplified with the help of PCR i.e. Polymerase chain reaction and multiple copies of desired DNA can be obtained to a level required for RFLP.

2] Restriction endonuclease: -

These are the enzyme which can cleave the DNA at a specific site present in the recognition sequence. This sequence may be 6-8 bp long and it is α palindromic sequence on the both the strand of DNA in a opposite direction. This enzyme is obtained from certain microorganism as these enzymes play a major role in the cell defense mechanism which bacteriophage enter the cell of these bacteria certain enzyme are produced with recognize some specific sequence on the phage DNA to cleave and destroy these DNA. Therefore, the entry of those phage is restricted. Therefore, these enzymes are called as Restriction enzyme/endonuclease.

3] Gel electrophoresis: -

The DNA fragment obtained the digestion with restriction enzyme can be separated on the basis of this length by gel electrophoresis. The fragment length solely depend on the frequency of recognition sequence and the sequence can be present on the DNA the various sites to the DNA is cleaved at every palindromic sequence and fragment are made.

4] Southern blotting: -

It is a technique by which the band obtained on agarose gel can be recorded permanently by transforming the band from gel to the membrane. A nylon/nitrocellular membrane is used for these purposes. Membrane is kept the agarose gel and top of it dry papers and heavy weight is pest. Due to the weight applied band start containing out of the gel and with the liquid part also start coming. The bands are trapped in the membrane and the liquid is soaked by the dry papers. This way the position of band on the membrane is exactly same as that on the gel.

5] Autoradiography: -

Radio labelled (⁸²P, ³³P,³⁵S and ³H) probe i.e. the ssDNA having ability to hybridize with the complimentary sequence are added on the membrane and allowed there to hybridize with the DN present on the bands under string out condition. After some time repeated washing is done to remove unbound probes and the membrane is held against the X-ray film, Radioactive compound in the bound probes emit rays which are captured by X-ray, and black-White image is formed when the image is developed the location of probes where they bind to the DNA on membrane is obtained.

Classical complement pathway, Alternative pathway, Lectin mediated pathway ?

2020-05-26T18:45:00.003+05:30

Classical complement pathway:

- after entry of antigen in the blood IgM and IgG type of antibody bind to its surface, other type of antibody’s [ IgD, IgE, IgA ] can not activate.

- Ones IgG and IgM antibody bind to surface of antigen conformation changes occur in Fc region of antibody bn Fc region of antibody due to this conformation changes

- A pocket is created for the binding of complement protein known as clqr2s2 after binding to Fc region of antibody clqr2s2 get activated and it gets photolytic activity clqr2s2 bar has to substrate

- C2 protein and C4 protein it clues C4 into C4a and C4b in which C4b is longer factor and C4a is smaller than smaller fragment dissociated and the larger fragment C4b bind to the surface of antigen it clues C2 into C2a and C2b in which C2a is longer fragment and C2b is smaller larger fragment C2a bind to C4b bond to antigen forming a dimer C4b2a.

- C4b2a bar gains the activity to degrade C3 protein therefore it is also known as C3 convertase clues 3a and C3b. C3a is smaller fragment which is dissociated in the serum C3b is larger fragment bind to C3a convertase (C3b2a) forming trimer C4b2a3b bar this trimer has ability to degrade C5 protein therefore it is known as C5 convertase.

- C5 convertase Can clues C5 protein into C5a and C5b is smaller fragment which is again dissociated and larger C5b protein sequence bind it to forming complex (C5bC6C7C8) C5b6789 complex has to ability gent inserted into membrane of antigen after getting the inserted into membrane multiple molecule of C9 protein bind to this complex forming C5b678 polynight this complex is also known as membrane attack complex which is form longer pore in the membrane of antigen and hence antigen is lised and killed.

Alternative Pathway: -

Antigen antibody complex for its activation the pathway starts with the autohydrolysis of complement protein C3 into C3a and C3b the rate of this autolytic reaction is very slow the C3a component is dissociated into serum while C3b which is larger factor it bind to it surface after its binding another serum protein joint to its which is known as factor B then factor D this complex and it clues factor B into fragment Ba and Bb, Ba is smaller factor which is dissociated Bb is larger which remain associated with C3b resulting in the dimer C3Bb factor D is dissociated after the cleavage of factor B C3bBb bar has ability to degrade C3 and therefore it is known as C3 convertase of alternative pathway this C3 convertase it clues C3 complement protein into C3a and C3b, C3b joins the dimer resulting in the complex C3bBb this is complex is also known as C5 convertase it clues C5 protein into C5a and C5b the larger C5b bind to the surface of antigen C5b678 polynight (9) this complex is also known is known as MAC which is form larger pore in the membrane of antigen and hence antigen is killed.

Lectin mediated pathway: -

Many bacteria and viral antigen have carbohydrate residues present on their surface this carbohydrate can be specifically recognize by serum protein host serum protein known as lectin. Such serum protein are also involved in immune activation

For example: -In human serum a protein known as [MBL] mannose binding lectin. Mannose residue present and form antigen and activate complex system.

Antigen bearing mannose residue comes in the body serum protein known [MBL] bind to mannose residue then two serum protein bind to MBW called as MASP-1, MASP-2 [MBL associated serum protein 1 and 2] MASP 1 and 2 have 2 substrate C2 and C4 compliment protein they clues then C4a and C4b and C2a and C2b, C4b and C2a forms complex of surface on antigen which is also called as C3 convertase C4b2a bar gains the activity to degrade C3 protein therefore it is also known as C3 convertase clues 3a and C3b. C3a is smaller fragment which is dissociated in the serum C3b is larger fragment bind to C3a convertase (C3b2a) forming trimer C4b2a3b bar this trimer has ability to degrade C5 protein therefore it is known as C5 convertase. C5 convertase Can clues C5 protein into C5a and C5b is smaller fragment which is again dissociated and larger C5b protein sequence bind it to forming complex (C5bC6C7C8) C5b6789 complex has to ability gent inserted into membrane of antigen after getting the inserted into membrane multiple molecule of C9 protein bind to this complex forming C5b678 polynight this complex is also known as membrane attack complex which is form longer pore in the membrane of antigen and hence antigen is lised and killed

Designing probe ?

2020-05-24T14:04:00.005+05:30

Designing probe: -

1. A probe is a nucleic acid molecule (single stranded DNA or RNA) with a strong affinity with a specific target

2. Probe and target base sequence must be compliment to each other beet depending on the conditions the do not necessarily have to be complementary.

3. Probe design depends upon whether a gene probe an oligonucleotide probe is desired.

Gene Probe: -

Gene probe are generally longer than 500 bp and comprise all or most of the target gene. They can be generated in two ways.

Clone Gene Probe: -

- Clone probe are generally used when clone species is available or when DNA sequence is unknown must be done first in order to be map and sequence.

- It is usual to cut the gene with restriction enzyme and excise it from and agarose gel, although if the vector has no this might not be necessary.

PCR: -

- PCR is α powerful procedure for making gene probe because it is possible to amplify and label at the same time.

- Long starches of DNA using chromosomal or plasmid DNA template.

- Labelled nucleotide molecule in extension step.

- A considerable amount of time, can be sequence when the gene of interest is PCR amplified, for there is no need for restriction enzyme digestion, Electrophoresis, elusion of DNA fragment from vectors.

- However, if the PCR amplification gives non-specific bond that will be used as a probe.

Oligonucleotide Probe: -

- Most common oligo robe contain 18-30bp.

- But current synthesis allows efficient synthesis of probe containing at least 100bp.

- Oligo probe can match perfectly to its target sequence sufficiently long to allow the use of hybridization condition that that will present the hybridization to other closely related sequence.

- Making it possible to identify and detect DNA with slight differences in sequence with in highly conserved gene. The selection of oligo probe sequence can be done manually from a known gene sequence using following gaudiness.

Guidelines : -

- The probe length should between 18 to 50 bases longer probe with result longer hybridization time and low synthesis yield.

- The base composition should be 40 to 60% GC nonspecific hybridization may increase for GC ratio outside of this range.

- Be certain that no complementary regions may present within the probe. This may result the formation of hairpin structure that will inhibit hybridization with target.

- Avoid sequence containing long stretches of a single base

- Once a sequence making the above criteria has been identified computerized sequence analysis is highly recommended.

- The probe sequence should be compared with the sequence region or genome from which it was derived as well as to the reverse complement of the region.

- It homology to non-target regions are grater than 70% or more bases in a row are found that probe sequence should not be used.

Random prime labeling (Prime extension)

A: Double stranded DNA is denatured and annealed with random oligonucleotide primers. (the oligonucleotide serves as a primer for 5’ 3’ fragment of E. coli) DNA polymerase which synthesis labelled probe in the presence of DIG-UTP.

B: In PCR-DIG labeling DIG-UTP is denatured and annealed with random oligonucleotide primer for the 5’à3’ polymerase. (The know fragment of E. coli polymerase I ) Which synthesis a labelled probe in presence of labelled nucleotide precursor.

Yeast two hybrid assay ?

2020-05-23T15:03:00.002+05:30

Yeast two hybrid assay: -

Study interaction, detecting protein-protein interaction.

Principle of the two-hybrid: -

A] Standard model of transcription activator: -

- The DNA binding domain of an activator binds to an enhancer.

- The activating domain interacted with the based complex. Recruiting it the promoter

B] Two hybrid assay for protein-protein interaction using gene clone technique linked the gene one protein to the part of a gene encoding a DNA binding domain, to encode 1 hybrid protein, linked the gene for another protein to the part of the gene encoding a transcription activating domain to encode a two hybrid proteins are introduce into yeast cells bearing the appropriate promoter enhancer and reporter gene (laczin) the two hybrid protein can get together and serve as an indicator activated transcription produces a bond and reporter gene product which can be detected with colorimetric assay using X-gal. (Blue colour compound)

For Example: -

One hybrid protein contributes DNA bonding domain, and other contributes or transcription activity domain the two parts of activator are held together by the interaction between protein X and Y. if X and Y

Interact and X gal is use in the assay reporter protein gene product yeast cell will turn blue if X and Y do not interact no activator gene will occur in this case the yeast cell will remain while in the presence of X gal the gal four DNA binding domain and transcription activity possibility exist.

C] Two hybrid screen for a protein that interact with protein zinc yeast cell are transform in 2 plasmid encoding a DNA binding domain coupled to a bait protein the other is a cell of plasmid containing many cDNA coupled to the coding region for a transcription activating domain each of this encode a fusion protein containing the activator domain fuse to and unknown DNA product each yeast cell is transform with just one of this pray encoding plasmid but several of their product are show together here for convince.

Once pray protein interact with the bait protein Z this brings together the DNA binding protein and the transcription activity domain so they can activate reporter gene.

the yeast two-hybrid system for detecting protein-protein interaction. the target protein fused to DNA-binding domain (Bait) when this target protein bind another specialy designed protein the cell nucleus (prey) theire interaction brings together and get transcription activate and the switches on the transcriptional reporter gene.

https://www.youtube.com/watch?v=lxrbYJE8hNM&t=6s

Reference: -

Young KH (February 1998). "Yeast two-hybrid: so many interactions, (in) so little time". Biology of Reproduction. 58 (2): 302–11. doi:10.1095/biolreprod58.2.302. PMID 9475380.

^ Jump up to:^a ^b ^c ^d ^e ^f ^g ^h ⁱ ^j ^k ^l ^m ⁿ ^o ^p ^q ^r ^s ^t ^u ^v ^w ^x Joung JK, Ramm EI, Pabo CO (June 2000). "A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions". Proceedings of the National Academy of Sciences of the United States of America. 97 (13): 7382–7. Bibcode:2000PNAS...97.7382J. doi:10.1073/pnas.110149297. PMC 16554. PMID 10852947.
^ Jump up to:^a ^b ^c ^d ^e ^f ^g ^h ⁱ Hurt JA, Thibodeau SA, Hirsh AS, Pabo CO, Joung JK (October 2003). "Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection". Proceedings of the National Academy of Sciences of the United States of America. 100 (21): 12271–6. Bibcode:2003PNAS..10012271H. doi:10.1073/pnas.2135381100. PMC 218748. PMID 14527993.
^ Fields S, Song O (July 1989). "A novel genetic system to detect protein-protein interactions". Nature. 340 (6230): 245–6. Bibcode:1989Natur.340..245F. doi:10.1038/340245a0. PMID 2547163. Abstract is free; full-text article is not.
^ Jump up to:^a ^b ^c Luo Y, Batalao A, Zhou H, Zhu L (February 1997). "Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system" (PDF). BioTechniques. 22(2): 350–2. doi:10.2144/97222pf02. PMID 9043710. Archived from the original (PDF) on 27 April 2017. Retrieved 26 April 2017.
^ Verschure PJ, Visser AE, Rots MG (2006). Step out of the groove: epigenetic gene control systems and engineered transcription factors. Advances in Genetics. 56. pp. 163–204. doi:10.1016/S0065-2660(06)56005-5. ISBN 9780120176564. PMID 16735158.^{[dead link]}
^ Jump up to:^a ^b Brückner A, Polge C, Lentze N, Auerbach D, Schlattner U (June 2009). "Yeast two-hybrid, a powerful tool for systems biology". International Journal of Molecular Sciences. 10(6): 2763–88. doi:10.3390/ijms10062763. PMC 2705515. PMID 19582228.
^ Gietz RD, Triggs-Raine B, Robbins A, Graham KC, Woods RA (July 1997). "Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system". Molecular and Cellular Biochemistry. 172 (1–2): 67–79. doi:10.1023/A:1006859319926. PMID 9278233.
^ Jump up to:^a ^b ^c ^d Auerbach D, Stagljar I (2005). "Yeast Two-Hybrid Protein-Protein Interaction Networks". Proteomics and Protein-Protein Interactions. Protein Reviews. 3. pp. 19–31. doi:10.1007/0-387-24532-4_2. ISBN 978-0-387-24531-7.
^ Whipple FW (August 1998). "Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli". Nucleic Acids Research. 26 (16): 3700–6. doi:10.1093/nar/26.16.3700. PMC 147751. PMID 9685485.

Toll-like Receptor ?

2020-05-22T09:54:00.000+05:30

1. Toll like receptor are a type of receptor of inmate immunity.

2. They are also known as pattern recognition protein because they can recognise a specific pattern associated with pathogen which are known as pathogen associated molecular pattern.

3. This type of receptors is not generated by VI-VDI recombination like TCR and BCR.

4. It recognises following type of pattern associated with pathogen.

Example: -

Toll like receptor	Pattern associated mol. pattern
TLR-2	Cell wall compound of Gram-positive bacteria, LPS and yeast cell wall component.
TLR-3	ds RNA
TLR-4	LPS
TLR-5	Flagellin

Disease associated with TLR (TLR-4) septic shock syndrome: -

1. The specific shock syndrome generally results from LPS release from many Gram-negative bacteria.

2. The pathophysiology is very complex and not completely understood.

3. The people at with developing septic shock including those who are hospitalise with chronic illness and poor nutritional status.

4. The risk is highest after the invasive procedure like subquery it also develops in immune compromised adults.

5. The septic shock begins with infection of pathogen in the blood.

6. Once the pathogen appear in the blood their LPS is released, release of LPS occur during cell division of bacteria and also upon its killing. (LbP)

7. In the blood lipid and part of LPS binds to lipid binding protein

8. To the lipid A complex of LPS is then transfer from LbP to CD₁₄ present on mΦ and dendritic cells.

9. CD₁₄then transfer lipid A to MD₂ (myeloid differentiating protein 2) after binding to lipid A Md₂ undergoes conformational changes and leads activation and dimerization of TLR-4

10. Activated TLR-4 induce activation of signal transducton pathway in which a transcription factor NFKB is activated.

11. NFKB helps in expression of gene of proinflammatory cytokines which induces TNF-α, IL and TL6 those cytokines affect vascular coagulation and inflammatory response which ultimately leads to septic shock.

12. The combine effect of this cytokine is widespread vasodilation, myocardial pump failure and multiorgan failure

13. The overall signal are as follows-

Signal: -

Hypotension (low BP)

Rapid respiration (high pulse rate)

Skin-pales and cold

Diarrhea

Wild spread blood clotting

High WBC count

Capillary leakage _ Abnormal body temperature

Tissue injury

Lethal organ failure

Treatment: -

Neutralization of TNF and IL with the help of monoclonal antibody and use of antagonistic which prevent fetial shock development.

Reference: -

1. Mahla RS, Reddy MC, Prasad DV, Kumar H (September 2013). "Sweeten PAMPs: Role of Sugar Complexed PAMPs in Innate Immunity and Vaccine Biology". Frontiers in Immunology. 4: 248. doi:10.3389/fimmu.2013.00248. PMC 3759294. PMID 24032031.

2. ^ Jump up to:^a ^b Hansson GK, Edfeldt K (June 2005). "Toll to be paid at the gateway to the vessel wall". Arteriosclerosis, Thrombosis, and Vascular Biology. 25 (6): 1085–7. doi:10.1161/01.ATV.0000168894.43759.47. PMID 15923538.

3. ^ Delneste Y, Beauvillain C, Jeannin P (January 2007). "[Innate immunity: structure and function of TLRs]". Médecine/Sciences. 23 (1): 67–73. doi:10.1051/medsci/200723167. PMID 17212934.

4. ^ Takeda K, Akira S (January 2005). "Toll-like receptors in innate immunity". International Immunology. 17 (1): 1–14. doi:10.1093/intimm/dxh186. PMID 15585605.

5. ^ Jump up to:^a ^b ^c Medzhitov R, Preston-Hurlburt P, Janeway CA (July 1997). "A human homologue of the Drosophila Toll protein signals activation of adaptive immunity". Nature. 388 (6640): 394–7. Bibcode:1997Natur.388..394M. doi:10.1038/41131. PMID 9237759.

6. ^ Jump up to:^a ^b Sharma N, Akhade AS, Qadri A (April 2013). "Sphingosine-1-phosphate suppresses TLR-induced CXCL8 secretion from human T cells". Journal of Leukocyte Biology. 93 (4): 521–8. doi:10.1189/jlb.0712328. PMID 23345392.

7. ^ Rolls A, Shechter R, London A, Ziv Y, Ronen A, Levy R, Schwartz M (September 2007). "Toll-like receptors modulate adult hippocampal neurogenesis". Nature Cell Biology. 9 (9): 1081–8. doi:10.1038/ncb1629. PMID 17704767.

8. ^ Du X, Poltorak A, Wei Y, Beutler B (September 2000). "Three novel mammalian toll-like receptors: gene structure, expression, and evolution". European Cytokine Network. 11(3): 362–71. PMID 11022119.

9. ^ Chuang TH, Ulevitch RJ (September 2000). "Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9". European Cytokine Network. 11 (3): 372–8. PMID 11022120.

10. ^ Tabeta K, Georgel P, Janssen E, Du X, Hoebe K, Crozat K, et al. (March 2004). "Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection". Proceedings of the National Academy of Sciences of the United States of America. 101(10): 3516–21. Bibcode:2004PNAS..101.3516T. doi:10.1073/pnas.0400525101. PMC 373494. PMID 14993594.

G-protein coupled receptors ?

2020-05-21T12:08:00.000+05:30

1. G-protein coupled receptor are type of receptor are types of receptor present on different kind of cell, this receptor have complex G-protein associated with its cytoplasmic tail.

2. G- protein receptor, are the protein which have the ability to exchange from GDP to GTP

3. Most of the G-protein are in active from when they are bound to GTP and inactive when they are bound to GDP.

4. One of the examples of such G-protein couple is epinaflen.

5. In this case there is a receptor for epinaflene on the cell surface and a complex of there G- protein at cytoplasmic region.

6. This protein are α, β, γ, G-protein.

7. When signal molecule binds to the receptor the α G-protein dissociate from β and γ with the exchange of GDP-GTP.

8. This dissociated GTP bound α-G protein then binds to mem-bound enzyme adenylate cyclase.

9. Adenylase cyclase is inactive without associated of GTP bound α-G protein.

10. When α-G protein bound to, it gets activated

11. After activation it catalases reaction in which ATP is converted into cyclic AMP.

12. Eventually GTP of α-G protein is hydrolyzed GDP. And due to this α-G protein dissociated from adenylate cyclase.

13. After this α-G protein rebound γ and β complex and ready for next cycle.

Pharmacogenomics

2020-05-21T10:14:00.003+05:30

Pharmacogenomics is the study of the role of the genome in drug response.

-Pharmacogenomics is the study how an individual genetic inheritance affected the body response to the drug.

-Pharmacogenomics aims to develop rational drug with respect to patients genotype to ensure maximum efficiency with minimal adverse effect.

-Taking into account the variability in the drug response from person to person, pharma-cogenesis ends at one drug for each individual [one drug for a group of persons with similar properties i.e. identical agent]

-Therefore, a better effect of the drug will be achieve preventing the adverse drug reaction.

Traditional drug designing ends at one for all approaches and the does depend on majority an age factor i.e low dose for young once and high dose for adults.

-But such problem can be illuminated by one person is equal to one drug approaches.

Four Steps in Pharmacokinetics [ADME]

1. Absorption.

2. Distribution.

3. Metabolism.

4. Excretion.

SNP: - Single Nucleotide Polymorphism.

Enzyme present in liver [at the side of metabolism]

e.g. Cytochrome, P450, TPMT, VKORC

In case of active drug: -

1. Enzyme activity high, drug dose is high.

2. Enzyme activity high, drug dose is low.

In case of inactive drug.

3. Enzyme activity high, drug dose is low.

4. Enzyme activity low, drug dose is high.

- All the drugs we inject are metabolism by our body.

- some drugs get activated after metabolism and some remain active before metabolism.

- This metabolism is carried out by special enzyme, which are generally present in liver cells.

e.g. Cytochrome P450

- This enzyme are expressed whenever they are required for the expression are not identical in every individual.

-Each gene sequence varies approximately nucleotide 100-300 bases. This is called as single, nucleotide polymorphism [SNP]

- Because the genes are different in their sequence the enzyme produced by expression of this gene also very in their activity from person to person.

- Therefore, the role of metabolism of a drug in each individual may differ

e.g. In case of drug which get inactivated after metabolism may show different response in different individual having different enzyme different individual having different enzyme activities.

1. In person having highly active enzyme the drug will be metabolised very fast and excreted from the body may not rich which is required to excretes its therapeutic effect.

2. In case of person having less enzyme activity the drug will be metabolise slowly, therefore it may remain in active from for a long period in the body.

3. The person with very slow metabolic rate may show toxic effect of that drug [e.g. in case of cytotoxic drug used in cancer therapy]

Benefit of Pharmacogenomics: -

1. Production of powerful medicine; -

2. Accuracy of therapy will be increased.

3. The undesirable side effect will be minimized and the patient will be prescribed drug based on their genotypes.

Disadvantages: -

1. It is not possible to study genotype of each individual.

2. One drug one-person approach is very costly.

3. It is not yet in use.

REFERENCE: -

1. Ermak, Gennady (2015). Emerging Medical Technologies. World Scientific. ISBN 978-981-4675-80-2.

2. ^ Jump up to:^a ^b Johnson JA (November 2003). "Pharmacogenetics: potential for individualized drug therapy through genetics". Trends Genet. 19 (11): 660–6. doi:10.1016/j.tig.2003.09.008. PMID 14585618.

3. ^ "Center for Pharmacogenomics and Individualized Therapy". Retrieved 2014-06-25.

4. ^ "overview of pharmacogenomics". Up-to-Date. May 16, 2014. Retrieved 2014-06-25.

5. ^ Jump up to:^a ^b Sheffield LJ, Phillimore HE (2009). "Clinical use of pharmacogenomic tests in 2009". Clin Biochem Rev. 30 (2): 55–65. PMC 2702214. PMID 19565025.

6. ^ Shin J, Kayser SR, Langaee TY (April 2009). "Pharmacogenetics: from discovery to patient care". Am J Health Syst Pharm. 66 (7): 625–37. doi:10.2146/ajhp080170. PMID 19299369.

7. ^ Jump up to:^a ^b "Center for Genetics Education".^[^{permanent dead link}^]

8. ^ Becquemont L (June 2009). "Pharmacogenomics of adverse drug reactions: practical applications and perspectives". Pharmacogenomics. 10 (6): 961–9. doi:10.2217/pgs.09.37. PMID 19530963.

9. ^ Hauser AS, Chavali S, Masuho I, Jahn LJ, Martemyanov KA, Gloriam DE, Babu MM (January 2018). "Pharmacogenomics of GPCR Drug Targets". Cell. 172 (1–2): 41–54.e19. doi:10.1016/j.cell.2017.11.033. PMC 5766829. PMID 29249361.

10. ^ "Guidance for Industry Pharmacogenomic Data Submissions" (PDF). U.S. Food and Drug Administration. March 2005. Retrieved 2008-08-27.

11. ^ Squassina A, Manchia M, Manolopoulos VG, Artac M, Lappa-Manakou C, Karkabouna S, Mitropoulos K, Del Zompo M, Patrinos GP (August 2010). "Realities and expectations of pharmacogenomics and personalized medicine: impact of translating genetic knowledge into clinical practice". Pharmacogenomics. 11 (8): 1149–67. doi:10.2217/pgs.10.97. PMID 20712531.

12. ^ Jump up to:^a ^b ^c Huser V, Cimino JJ (2013). "Providing pharmacogenomics clinical decision support using whole genome sequencing data as input". AMIA Joint Summits on Translational Science Proceedings. AMIA Joint Summits on Translational Science. 2013: 81. PMID 24303303.

13. ^ Jump up to:^a ^b Pirmohamed M (2001). "Pharmacogenetics and pharmacogenomics". Br J Clin Pharmacol. 52 (4): 345–7. doi:10.1046/j.0306-5251.2001.01498.x. PMC 2014592. PMID 11678777.

14. ^ Jump up to:^a ^b Prasad K (2009). "Role of regulatory agencies in translating pharmacogenetics to the clinics". Clin Cases Miner Bone Metab. 6 (1): 29–34. PMC 2781218. PMID 22461095.

15. ^ Evans DA, Clarke CA (1961). "Pharmacogenetics". Br Med Bull. 17 (3): 234–40. doi:10.1093/oxfordjournals.bmb.a069915. PMID 13697554.

16. ^ Kalow W (2006). "Pharmacogenetics and pharmacogenomics: origin, status, and the hope for personalized medicine". Pharmacogenomics J. 6 (3): 162–5. doi:10.1038/sj.tpj.6500361. PMID 16415920.

17. ^ Vogel F. Moderne Probleme der Humangenetik. Ergeb Inn Med Kinderheilk 1959; 12: 52–125

Transgenic plant

2020-05-19T15:49:00.000+05:30

-The production of pharmaceutical protein using transgenic plants has also gained some attention over last

-The introduction of foreign genes into plant spp. can be undertaken by a no of which Agrobacterium aim based vector. mediated gene transfer is most commonly employed.

-upon infection a portion of agrobacterium Ti plasmid is trans-located to a plant cell and is integrated into a plant cell genome. using such approaches, a whole range of therapeutic protein have been expressed in plant tissue.

-Depending upon specific promoter used, expression can be achieved uniformly throughout a whole plant or can be limited e.g. to expression in plant seeds.

Some proteins of Potential/alcohol Therapeutic interact that have been expressed: -

Protein	Expressed in	Production level achieved
Erythropoietin	Tobacco	0.003% of total soluble plant protein
Human serum albumin	Potato	0.02% of soluble leaf protein
Glucocerebrosidase	Tobacco	0.1 of weight leaf
Interferon-α	Rice	Not listed
Interferon-β	Tobacco	0.000002%of fresh weight 250 ng/ml
GM-CSF	Tobacco	250 ng/ml
Hirudin	Cannolo	1.0% seed of weight
Hepatitis B surface antigen	Tobacco	0.0007% of soluble protein
Antibodies/antibody fragment	Tobacco	various

Advantages: -

-Plant are regarded as partially attractive recombinant protein producers for a no. of reasons.

-cost of plant cultivation is low.

-Harvest equipment/methodologies are inexpensive as well established.

-Easy of scale-up.

-Plant based system are free oh human pathogen (e.g. HIV)

Disadvantages: -

-variable/low expression levels sometimes achieved.

-Potential occurrence of post- translation gene slicing.

-Glycosylation pattern achieved differ significantly from native human protein glycosylation pattern.

-Seasonal/geographical nature of plant growth.

-for these reasons, as well as a fact that additional tried and tested expression system are already available, production of recombinant the therapeutic protein in transgenic plant system has not as yet impacted significantly on industry.

REFERENCE:-

ISAAA 2013 Annual Report Executive Summary, Global Status of Commercialized Biotech/GM Crops: 2013 ISAAA Brief 46-2013, Retrieved 6 August 2014
^ Jump up to:^a ^b Pellegrino, Elisa; Bedini, Stefano; Nuti, Marco; Ercoli, Laura (15 February 2018). "Impact of genetically engineered maize on agronomic, environmental and toxicological traits: a meta-analysis of 21 years of field data". Scientific Reports. 8 (1): 3113. Bibcode:2018NatSR...8.3113P. doi:10.1038/s41598-018-21284-2. ISSN 2045-2322. PMC 5814441. PMID 29449686.
^ Jump up to:^a ^b ^c Klümper, W; Qaim, M (2014). "A Meta-Analysis of the Impacts of Genetically Modified Crops". PLOS ONE. 9 (11): e111629. Bibcode:2014PLoSO...9k1629K. doi:10.1371/journal.pone.0111629. PMC 4218791. PMID 25365303.
^ Andrew Pollack for the New York Times. 13 April 2010 Study Says Overuse Threatens Gains From Modified Crops
^ Jump up to:^a ^b Perry, E. D; Ciliberto, F; Hennessy, D. A; Moschini, G (2016). "Genetically engineered crops and pesticide use in U.S. Maize and soybeans". Science Advances. 2 (8): e1600850. Bibcode:2016SciA....2E0850P. doi:10.1126/sciadv.1600850. PMC 5020710.

Yeast as a source of biopharmaceutical.

2020-05-18T17:36:00.001+05:30

yeast are eukaryotic single called microorganism classified as a member of the fungus kingdom. many type of yeast are used for making many type of yeast are used for making many brewers yeast in beer fermentation and yeast in wine fermentation. Naturally yeast are found in plant leaves, flowers, fruit and soil.

Yeast cell are produced bio-pharmaceutical proteins. yeast cell grow in simple media with the help of genetic engineering by using r DNA technology the bio-pharmaceuticals protein products are easily obtain from yeast.

EXAMPLE:-

Yeast Species Protein

S. Cerevisiae Hepatitis vaccines Insulin

P. pastaris Human serum albumin IFN-Alpha

H.polymorpha HBV vaccine

Y. lipolytica Poncrelipase

Advantages:-

1. Grow fastly and inexpensive media require for growth.

2. Simple fermentation process is available.

3. Long history of industry application.

4. Molecular biology of yeast is studied in details.

5. It shows past translation modification.

Disadvantages:-

1. Glycosylating heterologous human proteins, which are different from native protein.

2. Expression level is less yeast as compare to E. coli.

PROTOCOL:-

REFERENCE:-

1.Ahmad

Hirz

Pichler

Schwab

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Appl Microbiol Biotechnol

2014

5301

2. Alper

Fischer

Nevoigt

Stephanopoulos

Tuning genetic control through promoter engineering

P Natl Acad Sci USA

2005

102

12678

3. Amano

Chiba

Kasahara

et al.

Engineering of mucin-type human glycoproteins in yeast cells

P Natl Acad Sci USA

2008

105

3232

4. Aouida

Piatek

Bangarusamy

Mahfouz

Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae

Curr Genet

2014

5. Berlec

Strukelj

Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells

J Ind Microbiol Biotechnol

2013

257

6. Blount

Weenink

Vasylechko

Ellis

Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology

PLoS ONE

2012

e33279

Product system using fungi as a source of biopharmacuticals

2020-05-17T16:45:00.001+05:30

-The great diversity of molecules produced by filamentous fungi justifies the explanation of these organisms

-The particular the isolation and identification of producing endophytic fungi is a new and feasible appropriate to the production of the anti-plastic drug.

-Fungi have elicited interest as heterogeneous protein producer's many have long history of use in the production of various industry/enzymes, such as Alpha-amylase and glycogmylase.

-however, due to certain futures such as very fast and strong growth and the ability to release large amount of protein directly to the substrate, this platform is most widely for the production of recombinant protein suitable fermentation technology therefore already exists.

- It was reported that fungi such as Asprgillus niger were able to produce and secrete glucomylase as Tricoderma resei even of extracellular protein.

currently A. nidulans, A.niger, N.creasea and T.reasei are mainly as expression system for obtaining protein

(e.g-antibodies)

In general fungi are capable of high-level expression of varios protein, many of which they secrete into their extracellular media.

The extracellular production of bio-pharmaceuticals would be distinctly advantages in terms of subsequent downstream processing.

Fungi process also the ability to carry out past downstream processing.

Fungi process also the ability to carry out past translation modification

Example:-

ORGANISM COMPOUND

1. Aspergillus spp Pro-teases

2. Penicillium nalgiovense Amphotenicin B

3. Taxomyces androme Taxol

Advantages:-

1. High level expression of protein.

2.It shows translation modification.

3.Suitable fermentation technology exists.

4.Most of species show secretion of protein extracellular.

Disadvantages:-

1.Pattern of glycosylation achieves can, however, differ from typical pattern obtained when a glycoprotein is expressed in a mammalian cell line.

2.Most fungal strain also naturally produced significant quantities of extracellular pro-teases, which can potentially degrade this recombinant product.

3.Although researcher have produced by such means has thus for sought or-gained marketing approval.

Diphtheria Toxins

2020-05-16T14:34:00.002+05:30

-It is exotoxin which secreted by carynebacterium diphtheria.

-It was first exotoxin to be discovered in 1890.

-Carnybacterium diphtheria is Gram negative aerobic non-motile rod classified as action bacteria.

-Diphtheria is an upper respiratory track disease characterized by sour throat fever etc.

-And there is an adherent membrane, this membrane is called as pseudo-membrane, which is present on tonsil and nasal cavity.

STRUCTURE OF TOXIN:-

-Diphtheria toxin is also a typical A,B type of toxin.

-It is produced as single polypeptide containing two fragment A and B which are contacted by disulfied linkage.

-Fragment A is 21kda, it has enzymatic activity for inhibit of eukaryotic elongation factor[eEF2]

-Fragment B is 35kda it helps in recognition of host call receptor but it is not involves in killing of target cell.

-The toxin cause disease diphtheria in human by gaining into the cell cytoplasm and inhibiting its protein synthesis.

MECHANISM OF ACTION

-After production of its fragments B recognition specific receptor called as trans-membrane hairpin specific receptors which is present on susceptible cell surface.

-When the toxin bind to receptor it taken inside by the cell by the process of endocytosis.

-And the toxin resistant in endosome of the cell.

-In the cell [in endosome] V type of ATP pase lowers

-A conformation change takes place in acidified endosome which is generally in fragments.

-The enzymatically active fragment [fragment A] is trans-located in cytoplasm.

-This active fragment is an enzyme [NAD+ dependent dipthamide ADP-ribosytransferase]

-This enzyme inactive the eEF2 protein by ADP. ribosylating the dipthamide.

-eEF2 is helps in translocation during protein synthesis.

By blocking the eEF2 by ADP-ribosylating dipthamide the process of protein synthesis is stop.

-Due to inhibition of protein synthesis the cell is killed.

REFERENCE:-

TABLE 1. Bacterial virulence properties altered by bacteriophages from Patrick L. Wagner; Matthew K. Waldor (August 2002). "Bacteriophage Control of Bacterial Virulence". Infection and Immunity. 70 (8): 3985–3993. doi:10.1128/IAI.70.8.3985-3993.2002. PMC 128183. PMID 12117903.
^ Bell CE, Eisenberg D (1996). "Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide". Biochemistry. 35(4): 1137–1149. doi:10.1021/bi9520848. PMID 8573568.
^ Jump up to:^a ^b Murphy JR (1996). "Corynebacterium Diphtheriae: Diphtheria Toxin Production". In Baron S; et al. (eds.). Medical microbiology(4th ed.). Galveston, Texas: Univ. of Texas Medical Branch. ISBN 978-0-9631172-1-2. PMID 21413281.
^ Jump up to:^a ^b Choe S, Bennett MJ, Fujii G, Curmi PM, Kantardjieff KA, Collier RJ, Eisenberg D (May 1992). "The crystal structure of diphtheria toxin". Nature. 357 (6375): 216–22. doi:10.1038/357216a0. PMID 1589020.
^ Jump up to:^a ^b Bell CE, Eisenberg D (January 1997). "Crystal structure of nucleotide-free diphtheria toxin". Biochemistry. 36 (3): 481–8. CiteSeerX 10.1.1.432.7047. doi:10.1021/bi962214s. PMID 9012663.
^ Jump up to:^a ^b ^c Bennett MJ, Eisenberg D (September 1994). "Refined structure of monomeric diphtheria toxin at 2.3 A resolution". Protein Sci. 3 (9): 1464–75. doi:10.1002/pro.5560030912. PMC 2142954. PMID 7833808.
^ Jump up to:^a ^b ^c Bell CE, Eisenberg D (January 1996). "Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide". Biochemistry. 35 (4): 1137–49. doi:10.1021/bi9520848. PMID 8573568.
^ Bennett MJ, Choe S, Eisenberg D (September 1994). "Refined structure of dimeric diphtheria toxin at 2.0 A resolution". Protein Sci. 3 (9): 1444–63. doi:10.1002/pro.5560030911. PMC 2142933. PMID 7833807.
^ Pappenheimer A (1977). "Diphtheria toxin". Annu Rev Biochem. 46 (1): 69–94. doi:10.1146/annurev.bi.46.070177.000441. PMID 20040.
^ Enke, U (2015): 125 Jahre Diphtherieheilserum, Dtsch Arztebl 2015; 112(49): A-2088

Tetanus Toxins

2020-05-15T09:43:00.002+05:30

-Tetanus toxins produced by clostridium tetani which is strictly anaerobic bacteria.

-it is also called as Tetanospasmin.

-it is neurotoxin.

-it is encoded by a plasmid and is a protein of single polypeptide which is cleaved into two factor A and B.

Structure of Tetanus Toxin:-

-The molecular weight is 150kda.

-Tetanus toxin is made u of two subunit A and B.

-B chain is heavy chain having weight 100kda and A chain is 50kda in weight

-B chain binds t gangliosides on the neuronal membrane and contain a translocation domain membrane and contains a translocation domain. which the movement of protein across that membrane and into the neutron.

-The A-chain, a zinc endopeptidase, attacks the vesicle-associated membrane protein (vAmp)

Mechanism of Action:-

-The toxin is produced under anaerobic codition and locally.

-Once it produced get absorbent by two negatively charged presynaptic membrane.

-After the absorption, the B subunit bind to ganglosides of neuromembrane and enter via.receptor mediated endocytosis.

-The strain is transfered from/neuron to other and finally reaches to CNS.

-The final step phagocytic vocuole become acidified and A subunit is released through the channel of B subunit of in cytopasm.

- The inhibitory-neurons normally work by releasing.

-The inhibitory like GABA (gamma amino butyric acid)

-This inhibitory generally binds on motor neurons.

-Normally glycin from inhibitory neuron and inhibit the muscle contraction.

-this allows relaxation of muscle in the affected person

- The action of toxin stop the affected neurons from releasing excretory transmitters, by degrading the protein synaptobrerin, The combined consequence is dangerous over-activity in the muscle from the smallest sensory stimulate as the damping of motor reflexes is inhibited leading to generalized contraction of the muscle, called as tetanic spasm.

REFERENCE:-

"tetanospasmin" at Dorland's Medical Dictionary
^ "Pinkbook | Tetanus | Epidemiology of Vaccine Preventable Diseases | CDC". www.cdc.gov. Retrieved 2017-01-18.
^ "Toxin Table » Environmental Health & Safety » University of Florida". www.ehs.ufl.edu. Retrieved 2017-01-18.
^ "Botulism". World Health Organization. Retrieved 2017-01-18.
^ Willey, Joanne (2009). Prescott's Principles of Microbiology. New York City, NY: McGraw-Hill. p. 481. ISBN 978-0-07-337523-6.
^ Jump up to:^a ^b Farrar JJ; Yen LM; Cook T; Fairweather N; Binh N; Parry J; Parry CM (September 2000). "Tetanus". Journal of Neurology, Neurosurgery, and Psychiatry. 69 (3): 292–301. doi:10.1136/jnnp.69.3.292. PMC 1737078. PMID 10945801.
^ AU Lalli G, Gschmeissner S, Schiavo G (November 15, 2003). "Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons". Journal of Cell Science. 116 (22): 4639–50. doi:10.1242/jcs.00727. PMID 14576357.
^ Eisel U, Jarausch W, Goretzki K, Henschen A, Engels J, Weller U, Hudel M, Habermann E, Niemann H (1986). "Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins". EMBO J. 5 (10): 2495–502. doi:10.1002/j.1460-2075.1986.tb04527.x. PMC 1167145. PMID 3536478.

High Throughput Screening

2020-05-14T21:22:00.004+05:30

1.HTS(high throughput screening) used for scientific experimentation especially used in drug discovery and relevant to the field of biology and chemistry.

2.using robotics data processing/ control software liquid handling device and sensitive detector.

3.HTS allows a researcher to quickly conduct millions of chemical, genetic or pharmacological tech.

4.through this process one can rapidly identify, active compound, antibodies or genes the modulate a particular bio molecule pathaway .

5.the result of these experiment provide starting points for drug design and for understanding the non interaction or role of a particular location.

Protocol for performing HTS assay:

1. Microtiter plate:- a small container, usually disposable and made of plastic, that contain small wells generally microtiter plate for HTS have either 96,192,384,1536,3456 or 6144 wells.

2.wells of microtiter plate usually act as reaction center for sample and reagent

3.well size of microtiter plate is very small and required very small amount of sample and reagent (either in µ/or nl )

4.Robot arm- for the addition of sample and reagent the robot arm join with multiple unit is used. which is automatically add accurate amount of sample add reagent at a time in multiple used. after addition then this microtiter plate is kept for incubation.

Obervation and Result:-

1.for the observation in wells the particular wells contain enzyme or cell and reagent either show observance or the chromagenic substrate is labelled to visualize reaction to give positive result.

2.generally plate reader is used for the assay of particular sample.

3.a plate reader having capacity to give accurate result at a time.

4.it gives all the result automatically after placing the microtiter plate in plate reader.

5.the result will display on computer software

Advantaged of HTS:-

1.fully automated system.

2.reduce error.

3.require very less amount of sample and reagent.

4.give accurate result within a very short time.

REFERENCE:-

Inglese J and Auld DS. (2009) Application of High Throughput Screening (HTS) Techniques: Applications in Chemical Biology in Wiley Encyclopedia of Chemical Biology (Wiley & Sons, Inc., Hoboken, NJ) Vol 2, pp 260–274 doi/10.1002/9780470048672.wecb223.
^ Macarron, R.; Banks, M.N.; Bojanic, D.; Burns, D.J.; Cirovic, D.A.; Garyantes, T.; Green, D.V.; Hertzberg, R.P.; Janzen, W.P.; Paslay, J.W.; Schopfer, U.; Sittampalam, G.S. (2011). "Impact of high-throughput screening in biomedical research". Nat Rev Drug Discov. 10 (3): 188–195. doi:10.1038/nrd3368. PMID 21358738.
^ Hann MM, Oprea TI (June 2004). "Pursuing the leadlikeness concept in pharmaceutical research". Curr Opin Chem Biol. 8 (3): 255–63. doi:10.1016/j.cbpa.2004.04.003. PMID 15183323.