Role of CRISPR/Cas9 in plant pathology
Production of disease resistance cultivars by editing the genome which is responsible for susceptibility factor for fungal and bacterial diseases.
By editing the genome which governs host pathogen interaction we can obtain incompatible interaction between host pathogen.
To improve the efficacy of bio control agents.
By editing the genome responsible for virus multiplication and virulence we can obtain virus free resistance cultivars.
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Akshay Deshmukh
clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Akshay Deshmukh
clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
Engineering plant immunity using crispr cas9 to generate virus resistanceSheikh Mansoor
Targeted genome editing by use of artificial nucleases has the plausible potential to speed basic research as well as plant breeding by providing the means to modify genomes quickly in a specific and predictable manner but advanced CRISPR-Cas9 based technologies first confirmed in mammalian cell systems are quickly being fitted for use in plants. These new technologies increase CRISPR-Cas9’s utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. RNA-guided genome editing using Streptococcus pyogenes CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) has renewed the concept of genome editing in plants. CRISPR-associated surveillance complexes are easily programmable molecular sleds that can target any sequence of choice. These complexes offer new opportunities for implementation in biotechnology. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. The CRISPR/Cas9 platform could also be used for targeted mutagenesis to identify host factors that control plant resistance and susceptibility to viral infection. Thus, CRISPR/Cas9 technology offers a promising approach for under- standing and engineering resistance to single and multiple viral infections in plants.
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
An Introduction to Crispr Genome Editing
Crispr cas: A new tool of genome editing
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are part of an adaptive defense mechanism in bacteria and archaea. Use of the CRISPR/Cas9 system for genome editing has been a major technological breakthrough, making genome modification in cells or organisms fast, more efficient, and much more robust than previous genome editing methods. Single guide RNAs (sgRNAs) or guide RNAs (gRNAs) direct and activate the Cas9 endonuclease at a specific genomic sequence. Cas9 then cleaves the target DNA, making it available for repair by the non-homologous end joining (NHEJ) system or for creating an insertion site for exogenous donor DNA by homologous recombination.
Engineering plant immunity using crispr cas9 to generate virus resistanceSheikh Mansoor
Targeted genome editing by use of artificial nucleases has the plausible potential to speed basic research as well as plant breeding by providing the means to modify genomes quickly in a specific and predictable manner but advanced CRISPR-Cas9 based technologies first confirmed in mammalian cell systems are quickly being fitted for use in plants. These new technologies increase CRISPR-Cas9’s utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. RNA-guided genome editing using Streptococcus pyogenes CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) has renewed the concept of genome editing in plants. CRISPR-associated surveillance complexes are easily programmable molecular sleds that can target any sequence of choice. These complexes offer new opportunities for implementation in biotechnology. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. The CRISPR/Cas9 platform could also be used for targeted mutagenesis to identify host factors that control plant resistance and susceptibility to viral infection. Thus, CRISPR/Cas9 technology offers a promising approach for under- standing and engineering resistance to single and multiple viral infections in plants.
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
An Introduction to Crispr Genome Editing
Crispr cas: A new tool of genome editing
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are part of an adaptive defense mechanism in bacteria and archaea. Use of the CRISPR/Cas9 system for genome editing has been a major technological breakthrough, making genome modification in cells or organisms fast, more efficient, and much more robust than previous genome editing methods. Single guide RNAs (sgRNAs) or guide RNAs (gRNAs) direct and activate the Cas9 endonuclease at a specific genomic sequence. Cas9 then cleaves the target DNA, making it available for repair by the non-homologous end joining (NHEJ) system or for creating an insertion site for exogenous donor DNA by homologous recombination.
CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.
Cas9 (CRISPR-associated protein 9) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
CRISPR : CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT
It is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity.
It forms the basis of a genome editing technology known as CRISPR-Cas9 that allows permanent modifications of genes within organisms.
CRISPR-Cas system consist of two key molecules that introduce a change into the DNA sequence 1. Cas 9 - act as molecular scissors 2. gRNA – guides Cas9 to the right part of the genome gRNA = crispr rRNA + tracrRNA
Prezi Link: https://prezi.com/q8lkxnmwk25-/untitled-prezi/?utm_campaign=share&utm_medium=copy
CRISPR is one of the mind blowing discovery which completely change the science of microorganisms. It is am efficient tool for genome editing and make the scientist enable to treat disease. The vast application of CRISPR technology covered almost all every aspect of life ranging from individual life to commercial aspect.
Purpose:
The purpose of this webinar is to develop creative scientific thinking in youngster and make them familiar with the miricals of science discovery.
CRISPR cas9 technology is a genome editing technique which won the noble prize in 2021.
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.
Genetic Engineering, Gene editing, Advantages of CRISPR, Limitations of CRISPR and Applications of CRISPR,
Introduction, History, components, cas9 protein structure and function,gRNA variants, Cas9 nuclease variants, CRISPR in bacteria as the immune system, mechanism, steps of working, Applications, and pros and cons.
Remote Sensing - A tool of plant disease managementAnand Choudhary
Definition, history , type of remote sensing, plateform used in remote sensing, type of resolution used in sensor, objective of remote sensing in plant disease management
All plant growth hormone like auxins cytokinin IBA ethylene and all hormone that are used in agriculture and horticulture purpose and useful for agriculture students for presentation purpose
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
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The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
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• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
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http://sandymillin.wordpress.com/iateflwebinar2024
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The Genome editing Era (CRISPER Cas 9) : State of the Art and Perspectives for the Management of Plant Diseases
1.
2. Seminar In-charge
Dr. Arjun Lal Yadav
Assistant Professor
Plant Pathology
Presented By
Anand Choudhary
Ph.D. Research Scholar
Plant Pathology
Department of plant Pathology
College of Agriculture, BiKaner
SKRAU, Bikaner (RAJ.)
3. Introduction
Importance of CRISPR Cas9 technique
Terminology
Origin of CRISPER Cas9 technique
Mechanism of CRISPER Cas9 technique
Uses of CRISPER Cas9 technique in plant pathology
Case studies of CRISPER Cas9 technique in plant disease management
Issues
Future prospect
4. CRISPER - Clustered Regularly Interspaced Short Palindromic Repeats.
Cas9 - CRISPR ASsociated protein 9
CRISPER Cas9- is a unique technology that enables geneticists and medical
researchers to edit parts of the genome by removing, adding or altering sections of
the DNA sequence.
It is currently the simplest, most versatile and precise method of genetic manipulation
and is therefore causing a buzz in the science world.
5. Importance
The CRISPR/Cas9 is considered a highly promising genome-editing method in crops because if its high degree
of flexibility and accuracy in cutting, multiple-gene editing, limited off-target impact, greater output and
simplicity
One of the reason for its popularity is that it makes is possible to carry out genetic engineering on an
unprecedented scale at a very low cost.
How it differs from previous technique, is that it allows for the introduction or removal of more than one
gene at a time, reducing the process from taking a number of year to a matter of weeks.
Its not species- specific, so can be used an organism previously resistant to genetic engineering.
In agriculture, it could help in the design of new grains, roots & fruits making the disease resistance cultivar.
Within the context of health it could pave the way to the development of new treatment for rare metabolic
disorders & genetic disease.
11. • Palindromic Sequence - A palindromic sequence is a nucleic acid
sequence in a double-stranded DNA or RNA molecule whereby reading in a
certain direction on one strand is identical to the sequence in the same
direction on the complementary strand.
12. Genome - A genome is the complete set of genetic information in an organism.
It provides all the information which the organism requires to function. In living organisms, the
genome is stored in long molecules of DNA called chromosomes.
The study and analysis of genomes is called genomics.
13. • Spacer DNA – Non coding DNA that separates one
gene from another. Spacers are short segments (26 to
72 bp) of sequence that are homologous to phage or
plasmid DNA.
• Cr RNA / CRISPER RNA- The crRNA is
complementary to the viral spacer that was stored after
the original infection.
• tracrRNA – Trans active RNA that bind with crRNA
form active complex.
• Sg RNA- Single guide RNA is a combination of tracer
RNA & cr RNA.
14.
15. • PAM sequence - Each Cas nuclease binds
to its target sequence only in presence of a
specific sequence, called protospacer
adjacent motif (PAM), on the non-targeted
DNA strand.
• The PAM is a component of the invading
virus or plasmid, but is not found in the
bacterial host genome
• The nuclease cuts 3-4 nucleotides upstream
of the PAM sequence.
21. In the last paragraph of the Discussion…..
This is how science often happens…….
22. In 1987 a Japanese team of scientists at osaka university noticed a new pattern of DNA sequence in a gene
belonging to E.coil
It appeared that the gene had five short repeating segments of DNA separated by short non spacer DNA
sequence. All five repeating segments had identical sequences composed of 29 bases, the building block of
DNA. By contrast each of the spacer sequence had their own unique sequence composed of 32 bases.
Microbiologists had never seen such a pattern before. By end of the 1990s however they had begun to
discover, with the aid new improvement of DNA sequencing.
That this pattern was prevalent in many different microbe species so common was the pattern that it was given
its own name, clustered regularly inter spaced short palindromic repeat
24. • In 2002, noted that another set of sequence always accompanied the CRISPR sequence. The Cas genes appeared
to code for enzyme that cut DNA.
• By 2005 three scientific teams had independently worked out that the spacer. Sequence between the crispr
sequence shared similarities with the DNA of viruses and hypothesised that it could be tool in the defense
mechanism of bacteria.
• Knowledge about how the CRISPR Cas9 system worked was opened up by some experiments conducted in 2007
by scientists at Danisco.
• The team infected a milk fermenting microbe Streptococcuss thermophilius, with two virus strains. Many of them
bacteria were killed by the virus, but some survived and went on to produce offspring also resistant to the viruses.
On further investigation it appeared that the microbes were inserting DNA fragment from the virus into their
spacer sequence and they lost resistance whenever new spacer were cut out.
25. • In Aug 2012, a small team of scientists led by Dr.
Jennifer Doudna (Right in image) and Dr. Emmanuel
Charpentier (left in image) published a paper in the
Science Journal, showing how to harness the natural
crispr cas9 system as a tool to cut any DNA strand in a
test tube.
• It wasn’t until 2020 - well after it had been adopted in
labs around the world - that Doudna and Charpentier
won the Nobel prize in Chemistry for their discovery,
becoming the first all-female team to do so.
26.
27. CRISPR Mechanism: How Does It Work?
The CRISPR-Cas9 system consists of two key molecules that introduce a change
(mutation) into the DNA. These are:
I. An enzyme called Cas9 which acts as a pair of molecular scissors that can cut the two
strands of DNA at a specific location in the genome so that bits of DNA can then be added
or removed.
II. A piece of RNA called guide RNA (g RNA) that consists of a small piece of predesigned
RNA sequence (about 20 bases pair long) located within a longer RNA scaffold.
The pre-designed sequence guides' the Cas9 to the right part of the genome. This makes
sure that the Cas9 enzyme cuts at the right point in the genome.
At this stage the cell recognises that the DNA is damaged and tries to repair it.
Scientists can use the DNA repair machinery to introduce changes to one or more genes in
the genome of a cell of interest.
28. CRISPR Mechanism: How Does It Work?
• Step 1. FORMATION OF THE EDITING COMPLEX
Case 9 enzyme pair with guide RNA, which carriers a sequence matching that
of the target gene.
29. CRISPR Mechanism: How Does It Work?
Step 2. PAIRING WITH THE TARGET GENE
The complex (Case9 g RNA and the complimentary sequence) binds precisely
to the target gene in the genome at PAM sites such as NGG, respectively in the
homologue sequence of host gene.
30. CRISPR Mechanism: How Does It Work?
Step 3. CUTTING THE TARGET DNA
• The transcrRNA pairs with pre-crRNA sequence to generate double stranded
RNA followed by cleaving with RNase III which produces mature crRNA.
• Case9 enzyme cuts the target gene on the genome.
31. QUESTION: What happens after the cut?
ANSWER: The cell tries to repair the damage!
Two Options
-----------------------------------------------------------------------------------------------------------------------------------------
( Error-prone)
Non-Homologous End Joining
Gene Knock-Out
or
(Requires a homologous donor DNA)
Homology Directed Repair
Gene Replacement
Neither outcome warrants the use of the word “editing”.
ATTGCCAGTCAGATCAGAGGTAA CTTACGGTGCATGACATTACTAGT
TAACGGTCAGTCTAGTCTCCATT GAATGCCACGTACTGTAATGATCT
?
32. Non-Homologous End Joining
Random bases added or deleted
Knock out = disrupt gene
X
Error prone DNA repair (NHEJ)
X
knock out = disrupt gene
33. CRISPR gene knockout
NHEJ is error-prone, and it usually results in insertions and deletions (indels) in the region
being repaired. When indels occur within the coding region of a gene and result in a
frameshift mutation, the gene becomes non-functional. This is known as a gene knockout.
34. 2nd option -Homology Directed Repair
DNA repair using a template (HR)
Repair instructions
Cell uses template to repair DNA
Alter the sequence to change function
Alter sequence to change function
35. CRISPR Mechanism: How Does It Work?
Step 4. INSERTING A NEW GENE
• A short fragment of DNA or the desired gene with a specific function is then
inserted to fill the gap and replace the original gene.
36. CRISPR knock-in
In the presence of a homology directed repair (DSB) induced by Cas9, cells can also repair
themselves via HDR, and this pathway offers an opportunity for researchers to insert a new
piece of DNA or an entire gene. This method is known as a gene knock-in.
37. CRISPR Mechanism: How Does It Work?
Step 5. PRODUCTION OF DESIRED PROTEIN
• The new gene is now ready to produce the desired protein in the cell or in a
test tube.
38. Role of CRISPR/Cas9 in plant pathology
Production of disease resistance cultivars by editing the genome which is
responsible for susceptibility factor for fungal and bacterial diseases.
By editing the genome which governs host pathogen interaction we can
obtain incompatible interaction between host pathogen.
To improve the efficacy of bio control agents.
By editing the genome responsible for virus multiplication and virulence
we can obtain virus free resistance cultivars.
39. The Genome-editing Era: State of The Art and Perspectives for the
Management of Plant Diseases
There are several strategies for researching plant disease resistance via the CRISPR/Cas
system:
i. knock-out of susceptibility factor encoding genes.
ii. deletion, modification, or introduction of cis-elements in promoters .
iii. introducing specific mutations in coding regions.
iv. alteration of amino acids in plant surface receptor proteins for evasion of secreted pathogen
effectors.
v. knock-out of negative regulators of plant defence responses.
vi. modification of central regulators of defense response .
40. Virus Resistance via CRISPR/Cas
The virus genome is replicated through a rolling-circle amplification mechanism via a dsDNA replicative form (Hanley-
Bowdoin et al., 2013).
Two recent works have also employed a CRISPR/Cas9 approach for achieving resistance to begomoviruses (Ali et al., 2015,
2016). The strategy of expressing the CRISPR/Cas9 system in the host cell nucleus to target and cleave the virus genome
during replication.
Protection against RNA viruses has seemed more difficult to achieve, since the classical SpCas9 from Streptococcus pyogenes
only recognizes dsDNA. However, the search for and characterization of related nucleases has led to the discovery of enzymes
that can bind to and cut RNA, such as FnCas9 from Francisella novicida.
The researchers have generated CRISPR mediated editing of host susceptible genes for developing viral resistance in plants. The
viral protein of potyviruses directly binds to eIF4E and completes its life cycle. Mutated eIF4E diminish the viral ability to
interact with host proteins and arrest the translation of the viral genome.
Site-specific DSB through CRISPR/Cas has opened up new dimension in targeting eIF4E for achieving complete resistance
against RNA based turnip mosaic virus (TuMV) in Arabidopsis (Pyott et al., 2016).
41. Resistance to Fungi Through CRISPR/Cas
Several strategies have been evolved to enhance fungal resistance in plant species based on the
current knowledge of molecular mechanisms implicated in plant-pathogen interaction. Potential
candidate genes and gene products involved in plant resistance against fungi have been described,
and nowadays these are prime targets for editing through the CRISPR/Cas9 approach.
Fister et al., (2018) reported for the first time the introduction of CRISPR/Cas9 components into
cacao leaves targeting the Non-Expressor of Pathogenesis-Related 3 (NPR3) gene, a suppressor
of the immune system, and obtained leaves with increased resistance to Phytophthora tropicalis.
1map mutants of F. graminearum showed two-fold reduction of mycotoxin production and were
unable to produce perithecia as well as to penetrate in wheat tissues (Urban et al., 2003).
48. • CRISPR mediated transgene free ‘Tomelo’ generated by deleting 48 bp
region from SlMLO1 locus and the resulted plants acquired resistance to
powdery mildew pathogen Oidium neolycopersici without affecting
phenotypic features and yield parameters (Nekrasov et al., 2017).
Leaves of tomato plants inoculated with Oidium neolycopersici (5 weeks post inoculation)
56. Fusarium head blight losses yield derives from sterility of infected florets,
grain quality reduction is mainly due to the accumulation of
trichothecenes—coded by the fungal tri genes cluster—highly toxic for
humans and animals.
In this studied, the author knocked-out 1tri5 and 1tri6 mutants of F.
graminearum were unable to spread the disease to the adjacent spikelets and
grains on wheat and corn, respectively, and also induced plant defense
responses.
57.
58. The technology faces two major issues
The first issues is a philosophical dilemma. Its centres on the extent to which CRISPR Cas
should be used to alter germ line cells eggs & sperm which is responsible for passing genes
to the next generation. While it will take many year before the technology will be viable to
use to create design babies. So great is the fear that some scientist, including some who
helped pioneers CRISPR Cas9, have called for a moratorium on its use in germ-line cells.
The second issue is one of safety. One of the major problems is that the technology is still
needs a lot of work to increase its accuracy and make sure that changes made in one part of
the genome do not introduce changes elsewhere which could have unforeseen
consequence.
59. Another critical issues is that once an organism such as a plant or insect, is
modified they are difficult to distinguish from the wild type and one released
into the environment could endanger biodiversity.
In another study, the CRISPR mediated MLO mutation in barley exhibited
resistance to powdery mildew (Blumeria graminis f. sp. hordei) but it
enhanced the susceptibility to rice blast fungus M. grisea.
60. Future prospects
In an era marked by political and societal pressure to reduce the use of pesticides, crop protection by genetic improvement
provides a promising alternative with no obvious impact on human health or the environment
The availability of novel or the improvement of known techniques that are safer for people and the environment is of
outmost importance to guarantee food safety and security especially in those countries where famine is still an important
issue (Vurro et al., 2010).
A novel technique that allows the production of precise knock-out mutants without the insertion of foreign DNA in a
saprotrophic/pathogenic fungus opens new possibilities of controlling plant pathogens.
The use of such edited fungal strains needs a correct strategy to minimize possible risks.
CRISPR tool can revolutionize the next generation agriculture by exploring the possibilities of the targeted crop species,
boost its resistance towards vulnerable pests, pathogens, consistency of productivity, abiotic stress tolerance, and enhance
nutritional efficiency (Ahmad et al., 2020). This technology may become next generation disease management tool for
sustainable crop improvement and next green revolution.
61. Acknowledgements
I would like to express my special thanks of gratitude to
Dr. A. L. Yadav Sir for their able guidance and support
and my classmate for provide internet service in complete
my presentation.
I gratefully acknowledge the use of some very important
information and photographs given in different review
paper written by Lander, E. S., 2016 ; Borrelli et al., 2018
and Munoz et al., 2019 and other researchers.
Editor's Notes
anand
Pus in an inflamed and infected area is due to a aggregation of neutrophils (white blood cells)
Fluid comes out of blood vessel, swelling, fluid uptake by lymphatic capillary to resolve swelling
Although there are many researcher who contributed to this researcher because research always doesn't work like one man Or women even two women enterprises - it is always builds up previous done research and it slowly slowly developed
Japanese team of scientists at osaka university noticed a new pattern of DNA sequence in a gene belonging to E.coil.