The dissertation analyzes the prevalence of the mecA gene in Methicillin-resistant Staphylococcus aureus (MRSA) isolated from clinical samples in Rajasthan. The objectives are to isolate MRSA from clinical samples using biochemical testing and the double disk synergy test, extract DNA from the isolates, and identify the presence of the mecA gene through PCR amplification and gel electrophoresis. Results showed the isolation of MRSA from various clinical samples and the presence of the mecA gene in some isolates.
Radial immunodiffusion (RID) or Mancini method is also known as Single radial immunodiffusion. An immunodiffusion technique, used in immunology to determine the quantity or concentration of an antigen in a sample.
Molecular analysis of plants transformed biolistically in general reveals a complex pattern of trans-gene, indicating the integration of multiple copies of the bombarded DNA.
Electroporation-A simple notable technique in our science field.Technique which is a magic of electric voltage.Technique that works on the mechanism of transient aqueous pore model.
DOI:10.21276/ijlssr.2016.2.4.5
ABSTRACT- Small Cardamom (Elettariacardamomum L. Maton) is one of the major spice crops of India, which were
the world’s largest producer and exporter of cardamom till 1980. There has however been a reduction in production,
mainly because of Katte disease, caused by cardamom mosaic virus (CdMV) a potyvirus. Viral diseases can be managed
effectively by early diagnosis using serological methods. In the present investigation, CdMV isolates were sampled from
Mudigere, Karnataka, ultra purified, and electron micro graphed for confirmation. Polyclonal antibodies were raised
against the virus and a direct antigen coating plate Enzyme linked immunosorbent Assay (DAC-ELISA) and Dot-ELISA
(DIBA) standardized to detect the virus in diseased and tissue cultured plants. Early diagnosis in planting material will aid
in using disease free material for better yields and hence increased profit to the farmer. Key-words- Cardamom mosaic virus (CdMV), Electron microscopy, Direct antigen coating Enzyme linked
immunosorbent Assay (DAC-ELISA), Dot-ELISA (DIBA)
Radial immunodiffusion (RID) or Mancini method is also known as Single radial immunodiffusion. An immunodiffusion technique, used in immunology to determine the quantity or concentration of an antigen in a sample.
Molecular analysis of plants transformed biolistically in general reveals a complex pattern of trans-gene, indicating the integration of multiple copies of the bombarded DNA.
Electroporation-A simple notable technique in our science field.Technique which is a magic of electric voltage.Technique that works on the mechanism of transient aqueous pore model.
DOI:10.21276/ijlssr.2016.2.4.5
ABSTRACT- Small Cardamom (Elettariacardamomum L. Maton) is one of the major spice crops of India, which were
the world’s largest producer and exporter of cardamom till 1980. There has however been a reduction in production,
mainly because of Katte disease, caused by cardamom mosaic virus (CdMV) a potyvirus. Viral diseases can be managed
effectively by early diagnosis using serological methods. In the present investigation, CdMV isolates were sampled from
Mudigere, Karnataka, ultra purified, and electron micro graphed for confirmation. Polyclonal antibodies were raised
against the virus and a direct antigen coating plate Enzyme linked immunosorbent Assay (DAC-ELISA) and Dot-ELISA
(DIBA) standardized to detect the virus in diseased and tissue cultured plants. Early diagnosis in planting material will aid
in using disease free material for better yields and hence increased profit to the farmer. Key-words- Cardamom mosaic virus (CdMV), Electron microscopy, Direct antigen coating Enzyme linked
immunosorbent Assay (DAC-ELISA), Dot-ELISA (DIBA)
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
TOXICOLOGY STUDY IS VERY ESSENTIAL FOR DRUG DISCOVERY. INTERNATIONAL COUNCIL FOR HARMONIZATION (ICH) HAS IMPLEMENTED SOME BASIC RULES AND REGULATION REGARDING THE TOXICITY STUDY ON ANIMAL DURING PRE-CLINICAL TRIAL, WHICH IS A PART OF DRUG DISCOVERY PROCESS. HERE SOME OF THE BASIC TEST ARE DISCUSSED ALONG WITH SOME BASIC CONCEPTS OF GENETICS. HOPE THIS WILL HELP THE STUDENTS TO UNDERSTAND THE TOPIC.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
2. DISSERTATION
ON
Prevalence of mecA gene in MRSA from Isolated
clinical samples of patients from Rajasthan
Submitted to Submitted By
Ms Apoorva Rana Oshin Raj
Assistant Research Scientist BSc Biotechnology
Dr. B Lal Institute of Biotechnology Part III
3. Introduction
• Staphylococcus aureus is a gram positive, cocci
shaped bacterium basically found on the surface of
the nose and ear and respiratory tract (Bata et.
al.;2014)
• They belong to the family of Firmicutes.
• S. aureus is not always pathogenic in nature,
commonly causes skin infection.
• Staphylococcus was first identified in 1880 by Sir
Alexander Ogston.
• S. aureus is catalase positive (that is it can produce
enzyme catalase).
• S. aureus was found to have natural genetic
transformation but only at low frequency.
4. Methicillin resistant Staphylococcus aureus
• Methicillin resistant Staphylococcus aureus (MRSA)
is bacterium that causes infections in different parts of
the body.
• Its tougher to treat than most Staphylococcus because it
is resistant to some commonly used antibiotics.
• The symptoms of MRSA depend on where you’re
infected. Most often, it cause mild infections on skin
but also cause more serious skin infections in lungs or
urinary tract.
• Staphylococcus can usually be treated with antibiotics.
But over decades, some strains have become resistant
to antibiotics that once destroyed it.
• It’s now resistant to methicillin, amoxcillin, penicillin,
oxacillin and other common antibiotics.
5. OBJECTIVES
• To analyse the prevalence of mecA gene responsible for
methicillin resistance in S. aureus (MRSA)
6. Work plan
Isolation and identification of Methicillin
Resistant Staphylococcus aureus (MRSA) from
clinical samples
DNA Extraction and its amplification
Molecular typing of the gene responsible for the
MRSA
7. Isolation and identification of Methicillin Resistant Staphylococcus aureus (MRSA)
from clinical samples
Isolation of bacterial isolates
from cinical samples of
infected patients by Streaking
Method
Identification of
Staphylococcus aureus by
Biochemical Testing
Identification of MRSA by
Double Disk Synergy Test
Molecular typing of the gene responsible for the resistance in Staphylococcus aureus
(MRSA)
METHODOLOGY
Extraction of DNA from
MRSA isolates
Amplification of DNA by
Polymerase Chain Reaction
against the targeted primers
Molecular Typing by
Agarose Gel
Electrophoresis to identify
the gene present
8. Isolation of S. aureus from clinical samples by streaking
Material and method
11. DNA Isolation
Culturing of bacteria in nutrient broth medium and incubation at 37°C over night with
shaking.
Transfer culture to 1.5 ml eppendroff tube and spin down cell culture at high speed for 1
minute at table top centrifuge.
Discard the supernatant to remove the liquid completely by mixing upside down onto a
piece of paper towel for a few second.
Add 100µl of lysis solution (P2 Buffer) and mix by gentle inverting the tubes 5-6 times.
The solution should quickly turn transparent and become more viscous indicating bacterial
lysis has taken place.
Add 100µl of re-suspension solution (P1 Buffer) into each tube and vortex to completely
re-suspend cell pellet.
Add 150 µl of neutralizing solution (P3 Buffer) and mix by inverting the tubes several
times. At this point bacterial chromosomal DNA is usually seen as white precipitate.
12. Centrifuge the tubes at high speed for 10 minutes. At 10000 rpm at 3°C.
Carefully transfer the supernatant to a new eppendorfs.
Add 2.5-3 volume of chilled ethanol to each tube and mix by inverting the tube a few
times.
Spin down plasmid DNA precipitate at high speed for 10 minutes at 10000 rpm at 3°C.
Discard the supernatant and remove the remaining liquid as much as possible by leaving
the tube upside down on a piece of paper towel. Then keep the tubes in a tube holder and
air dry for 10-20 minutes. To dry faster, keep tubes at 37°C heat blocker. DNA precipitate
turns white when dry.
Re-suspend the DNA pellet with 50µl TE Buffer. Completely dissolve the pellet by
pipetting solution several times
13. Qualitative and Quantitative estimation of
DNA
1O µl DNA sample was taken in TE Buffer
The sample was diluted by the factor of 100 i.e. by taking 10 µl of
sample in 990µl of TE Buffer
After dilution, the optical density value calculated the total amount of
DNA recovered
The concentration of DNA was determined by using the formula-
A260×50×dilution factor/1000
After finding the concentration of DNA, quality of DNA was calculated
by taking the ratio O.D at 260/280
14. Polymerase chain reaction (PCR) is a
technique used in molecular biology to
amplify a single copy or few copies of a
segment of DNA across several orders of
magnitude, generating thousands of millions
of copies of a particular DNA sequence. It is
an easy, cheap and reliable way to repeatedly
replicate a focused segment of DNA , a
concept which is applicable to numerous
field in modern biology and related sciences.
Polymerase chain reactions
15. Gel Electrophoresis
• Gel electrophoresis is a technique commonly used in laboratories to
separate charged particles like DNA, RNA, PROTEINS according to
their size.
• Charged molecules move through a gel when an electric current is
passed across it.
• An electric current is applied across the gel so that one end of the gel
has a positive charge and the other end has a negative charge.
• The gel consists of a permeable matrix, like a sieve, through which
molecules can travel when an electric current is passed across it.
• Smaller molecules migrate through the gel more quickly and
therefore travel further than larger fragments that migrate more
slowly and therefore will travel a shorter distance. As a result the
molecules are separated by size.
20. • Antibiotic sensitivity test was performed by the commonly used
Disc diffusion method which is designed to determine the
smallest amount of the antibiotic needed to inhibit the growth of a
microorganism.
• MRSA isolates was cultured on agar plates using streaking
method.
• DNA of the isolated sample was extracted using the crude boiling
method.
Conclusion
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