The yeast two-hybrid system is a technique used to study protein-protein interactions. It involves creating two fusion proteins, one with a DNA-binding domain and one with an activation domain. If the proteins of interest interact, they will reconstitute a functional transcription factor that activates a reporter gene. This allows researchers to identify novel interactions and characterize known interactions, helping to further understand biological processes at the molecular level.
A brief introduction to two techniques used to study protein interactions: Yeast two hybrid (Y2H) system and Chromatin immunoprecipitation(ChIP)
I hope it helps and please comment if I've made any mistakes.
The three hybrid system of yeast has been described in this ppt. Yeast one Hybrid system, yeast two hybrid system and yeast 3 hybrid system is explained. This explain about the DNA-protein interaction and Protein-DNA-Protein interaction.
Yeast two hybrid system for Protein Protein Interaction Studiesajithnandanam
Yeast Two Hybrid system uses a reporter gene to detect the interaction of pair of proteins inside the yeast cell nucleus. In the yeast Two Hybrid System, The interaction of target protein to the protein will bring together transcriptional activator, which then switches on the expression of reporter gene.
A brief introduction to two techniques used to study protein interactions: Yeast two hybrid (Y2H) system and Chromatin immunoprecipitation(ChIP)
I hope it helps and please comment if I've made any mistakes.
The three hybrid system of yeast has been described in this ppt. Yeast one Hybrid system, yeast two hybrid system and yeast 3 hybrid system is explained. This explain about the DNA-protein interaction and Protein-DNA-Protein interaction.
Yeast two hybrid system for Protein Protein Interaction Studiesajithnandanam
Yeast Two Hybrid system uses a reporter gene to detect the interaction of pair of proteins inside the yeast cell nucleus. In the yeast Two Hybrid System, The interaction of target protein to the protein will bring together transcriptional activator, which then switches on the expression of reporter gene.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
insilico protein structure prediction and and structure analysis and its type which are commonly used in dry lab. docking is a procedure in which two protein,or protein to ligand binding intrection analysis by software tools.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
insilico protein structure prediction and and structure analysis and its type which are commonly used in dry lab. docking is a procedure in which two protein,or protein to ligand binding intrection analysis by software tools.
Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.
Most bacteria are free-living organisms that grow by increasing
in mass and then divide by binary fission.
Growth and division are controlled by genes, the expression
of which must be regulated appropriately. Genes
whose activity is controlled in response to the needs of a
cell or organism are called regulated genes. All organisms
also have a large number of genes whose products
are essential to the normal functioning of a growing and
dividing cell, no matter what the conditions are. These
genes are always active in growing cells and are known as
constitutive genes or housekeeping genes; examples include
genes that code for the enzymes needed for protein
synthesis and glucose metabolism. Note that all genes are
regulated on some level. If normal cell function is impaired
for some reason, the expression of all genes, including
constitutive genes, is reduced by regulatory
mechanisms. Thus, the distinction between regulated
and constitutive genes is somewhat arbitrary.
1. Levels of gene regulation
The observation that differences in the RNA and protein content of different tissues are not paralleled by significant differences in their DNA content indicates that the process whereby DNA produces mRNA must be the level at which gene expression is regulated in eukaryotes. In bacteria this process involves only a single stage, that of transcription, in which RNA copy of the DNA is produced by the enzyme RNA polymerase. Even while this process is still occurring, ribosomes attach to the nascent RNA chain and begin to translate it into protein. Hence cases
of gene regulation in bacteria, such as the switching on of the synthesis of the enzyme β-galactosidase in response to the presence of lactose (its substrate), are mediated by increased transcription of the appropriate gene. Clearly, a similar regulation of gene transcription in different tissues, or in response to substances such as steroid hormones which induce the synthesis of new proteins, represents an attractive method of gene regulation in eukaryotes.
In contrast to the situation in bacteria, however, a number of stages intervene between the initial synthesis of the primary RNA transcript and the eventual production of mRNA (Fig. 1).
The initial transcript is modified at its 5′ end by the addition of a cap structure containing a modified guanosine residue and is subsequently cleaved near its 3′ end, followed by the addition of up to 200 adenosine residues in a process known as polyadenylation. Subsequently, intervening sequences or introns, which interrupt the protein-coding sequence in both the DNA and the primary transcript of many genes. Although this produces a functional mRNA, the spliced molecule must then be transported from the nucleus, where these processes occur, to the cytoplasm where it can be translated into protein.
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
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MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
2.
We know protiens are isolated entities
They diffuse freely for random collision of
protein A and B
But this picture is too simple to account for
complex processes that colesce to form life
Protein machines or aggragates of 10
machines carry out majority of cellular
phenomena
Yeast 2 hybrid system is a technique used
to study protein-protein interaction.
3. A
protein is composed of domains, which
allow the same protein to perform different
functions
The yeast two-hybrid technique uses two
protein domains that have specific functions:
1.
a DNA-binding domain (BD), that is capable
of binding to DNA,
2. an activation domain (AD), that is capable
of activating transcription of the DNA.
Both of these domains are required for
transcription.
4. DNA
mRNA
protein.
Transcription requires a protein called a
transcriptional activator (TA).
Promoter is a region situated upstream from
the gene coding for DNA.
TA bound to the promoter via binding domain
it activates transcription via its activation
domain.
If either of domains is absent, then
transcription of the gene will fail.
5. Figure 1. Normal Transcription. Normal transcription
requires both the DNA-binding domain (BD) and the
activation domain (AD) of a transcriptional activator (TA).
6.
1.
2.
-the binding domain and the activation domain do
not necessarily have to be on the same protein. In
fact, a protein with a DNA binding domain can
activate transcription when simply bound to another
protein containing an activation domaintwo fusion proteins are created:
protein of interest (X), it has DNA binding domain
attached to its N-terminus
potential binding partner (Y), which is fused to an
activation domain.
the binding of these two will form an intact and
functional transcriptional activator.
It transcribe a reporter gene, whose protein product
can be easily detected and measured
7. Figure 2. Yeast two-hybrid transcription. The yeast twohybrid technique measures protein-protein interactions by
measuring transcription of a reporter gene. If protein X and
protein Y interact, then their DNA-binding domain and
activation domain will combine to form a functional
transcriptional activator (TA). The TA will then proceed to
transcribe the reporter gene that is paired with its promoter.
8. construct
the ‘bait’ and ‘hunter’ fusion
proteins.
1. The ‘bait’ fusion protein is the protein of
interest linked to the GAL4 BD.
2. This plasmid will also have inserted in it a
segment of Gal4 BD DNA next to the site of
bait DNA insertion.
3. when the DNA from the plasmid is
transcribed and converted to protein, the
bait will now have a binding domain
attached to its end.
9. Figure 3. Plasmid construction. The ‘bait’ and ‘hunter’ fusion
proteins are constructed in the same manner. The ‘bait’ DNA is
isolated and inserted into a plasmid adjacent to the GAL4 BD DNA.
When this DNA is transcribed, the ‘bait’ protein will now contain the
GAL4 DNA-binding domain as well. The ‘hunter’ fusion protein
contains the GAL4 AD.
10. Figure 4. Bait and Hunter Plasmids. The yeast two-hybrid
assay uses two plasmid constructs: the bait plasmid, which is
the protein of interest fused to a GAL4 binding domain, and
the hunter plasmid, which is the potential binding partner
fused to a GAL4 activation domain.
11. Plasmids
are introduced into host cell by
transfection.
Isolate the colonies with both bait and
hunter on minimal media
The cells used for transfection are called
auxotrophic mutants
Cells containing the plasmid are able to
survive on the minimal media
Selection occurs in two rounds: first on one
minimal media plate, to select for the ‘bait’
plasmid, and then on another minimal media
plate, to select for the ‘hunter’
12. Once inside the cell, if binding occurs between
the hunter and the bait, transcriptional activity
will be restored and will produce normal Gal4
activity.
reporter gene used in the Gal4 system is LacZ,
an E. coli gene whose transcription causes cells
to turn blue
In this yeast system, the LacZ gene is inserted in
the yeast DNA immediately after the Gal4
promoter, so that if binding occurs, LacZ is
produced.
Therefore, detecting interactions between bait
and hunter simply requires identifying blue
versus non-blue
13. Figure 5. Transfection. The ‘bait’ and ‘hunter’ plasmids are introduced
into yeast cells by transfection. In this process, the plasma membrane is
disrupted to yield holes, through which the plasmids can enter. Once
transfection has occurred, cells containing both plasmids are selected for
by growing cells on minimal media. Only cells containing both plasmids
have both genes encoding for missing nutrients, and consequently, are
the only cells that will survive.
14. can identify novel protein-protein interactions.
By using a number of different proteins as
potential binding partners, it is possible to
detect interactions that were previously
uncharacterized
Secondly, the yeast two-hybrid assay can be used
to characterize interactions already known to
occur.
Characterization could include determining
which protein domains are responsible for the
interaction or under what conditions interactions
take place, by altering the intracellular
environment.
15.
application of the yeast two-hybrid involves
manipulating protein-protein interactions in an
attempt to understand its biological relevance.
For example, many disorders arise due to mutations
causing the protein to be non-functional, or have
altered function.
in some cancers; a mutation in a pro-growth
pathway does not allow for the binding of negative
regulatory proteins, resulting in the pathway never
turning ‘off’.
The yeast two-hybrid is one means of determining
how mutation affects a protein’s interaction with
other proteins.
When a mutation is identified that affects binding,
the significance of this mutation can be studied
further
16. elegant means of investigating protein-protein
interactions.
A fairly new addition to the family of
microbiological studies
understanding of biological systems in the past
few years.
While isolation of a protein in an attempt to
understand its function still has its place in
biological research
biological reactions do not occur in isolation. A
protein is constantly interacting with other
proteins in what we now know to be a delicate
balance.
17.
1. Alberts, B. (1998). The Cell as a Collection of
Protein Machines: Preparing the Next Generation
of Molecular Biologists. Cell 92: 291-4.
2. Fields S, Song O.K. (1989). A novel genetic
system to detect protein-protein interactions.
Nature 340:245-6.
3. Fields S, Sternglanz R. (1994). The two-hybrid
system: an assay for protein-protein
interactions. Trends in Genetics 10:286-92.
4. Two-hybrid analysis of genetic regulatory
networks — online protocol