This document summarizes the analysis of MHC haplotypes in two rare Greek sheep from the Argos breed. It describes the MHC structure and genes studied, including DRB1, DRA, DQA, DQB, and class I genes. Materials and methods for genotyping these genes using PCR and sequencing are provided. The results found new alleles for several MHC genes, indicating high diversity in this rare breed. Optimizing PCR cycles helped reduce hybridization and errors in class I genotyping. Overall, the study characterized MHC polymorphism in the endangered Argos breed.
Rice is the principal food crop for more than half of the
world's population. Rice, as a staple food, supports more
than three billion people and comprises 50%–80% of their
daily calorie intake [1]. Adverse environmental factors
such as excessive cold, heat, drought, and salinity stresses
result in a considerable yield loss of crop plants all over
the world. Plant adaptations to environmental stresses
depend on the activation of cascades of molecularnetworks involved in signal transduction, stress perception,
and expressions of stress‐related genes. These
abiotic stresses elicit complex cellular responses in the
plant system, resulting in the production of excessive
reactive oxygen species (ROS) such as hydrogen peroxide
(H2O2), hydroxyperoxyl (HO2·), superoxide (O2
−), and
singlet oxygen (1O2) radicals. To protect themselves from
adverse conditions, plants have evolved a number of
cellular defense mechanisms including antioxidants such
as ascorbate, glutathione, and tocopherols as well as
ROS‐detoxifying enzymes such as superoxide dismutases
(SODs), peroxidases, and catalases (CATs) [2,3].
Rice is the principal food crop for more than half of the
world's population. Rice, as a staple food, supports more
than three billion people and comprises 50%–80% of their
daily calorie intake [1]. Adverse environmental factors
such as excessive cold, heat, drought, and salinity stresses
result in a considerable yield loss of crop plants all over
the world. Plant adaptations to environmental stresses
depend on the activation of cascades of molecularnetworks involved in signal transduction, stress perception,
and expressions of stress‐related genes. These
abiotic stresses elicit complex cellular responses in the
plant system, resulting in the production of excessive
reactive oxygen species (ROS) such as hydrogen peroxide
(H2O2), hydroxyperoxyl (HO2·), superoxide (O2
−), and
singlet oxygen (1O2) radicals. To protect themselves from
adverse conditions, plants have evolved a number of
cellular defense mechanisms including antioxidants such
as ascorbate, glutathione, and tocopherols as well as
ROS‐detoxifying enzymes such as superoxide dismutases
(SODs), peroxidases, and catalases (CATs) [2,3].
Co-amplification at lower denaturation temperature PCR specifically used for precise detection of mutation in DNA samples.It also improves precision of downstream techniques used for mutation detection
Use of Automated High Content Analysis Applied To Assessment Of Primary DNA D...HCS Pharma
Quantifying DNA damage is mandatory to assess potential adverse effects of candidate, drugs, molecules or extracts developed in dermo-cosmetic industry. Different assays can be performed to detect primary DNA damages, such as γH2AX or the single cell gel electrophoresis also known as the comet assay. The High Content Imaging technology was used as a valuable tool for screening in early discovery phase.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
MilliporeSigma's Jennifer Pratt recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating the utility of HepaRG MRP2 Knockout cells for investigating drug-transporter interactions in the liver involving MRP2.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.
Probability Models for Estimating Haplotype Frequencies and Bayesian Survival...Université de Dschang
M. Kum Cletus Kwa a soutenu une thèse de Doctorat/Phd en mathématiques ce 14 juin 2016 à l'Université de Dschang. Le jury lui a décerné à l'issue des échanges la mention très honorable.
Co-amplification at lower denaturation temperature PCR specifically used for precise detection of mutation in DNA samples.It also improves precision of downstream techniques used for mutation detection
Use of Automated High Content Analysis Applied To Assessment Of Primary DNA D...HCS Pharma
Quantifying DNA damage is mandatory to assess potential adverse effects of candidate, drugs, molecules or extracts developed in dermo-cosmetic industry. Different assays can be performed to detect primary DNA damages, such as γH2AX or the single cell gel electrophoresis also known as the comet assay. The High Content Imaging technology was used as a valuable tool for screening in early discovery phase.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
MilliporeSigma's Jennifer Pratt recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating the utility of HepaRG MRP2 Knockout cells for investigating drug-transporter interactions in the liver involving MRP2.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.
Probability Models for Estimating Haplotype Frequencies and Bayesian Survival...Université de Dschang
M. Kum Cletus Kwa a soutenu une thèse de Doctorat/Phd en mathématiques ce 14 juin 2016 à l'Université de Dschang. Le jury lui a décerné à l'issue des échanges la mention très honorable.
An Overview...
Definition of Translation.
Def. of Eukaryotes.
Translation: An Overview.
Components of Translation.
Some Enzymes .
Ribosome Role.
Mechanism of Translation.
Initiation.
Scanning Model of Initiation.
Initiation Factors.
Animation.
Elongation.
Chain Elongation: Translocation.
Animation.
Termination.
Animation....
It's not perfect still... what are your views friends?
general information regarding single nucleotide polymorphism.
A Single Nucleotide Polymorphisms (SNP), pronounced “snip,” is a genetic variation when a single nucleotide (i.e., A, T, C, or G) is altered and kept through heredity.
In this research paper from the Spring 2015 semester, I described my analysis of certain genome scaffolds, or gaps within the Malaclemys terrapin genome. I examined seven of these scaffolds and determined their approximate sizes through Polymerase Chain Reaction (PCR) and Gel Electrophoresis. The DNA was then prepped to be sent for sequencing by an external source. The resulting chromatograms gave inconclusive results on the exact sequences of these scaffolds.
DETECTION OF BACTERIAL PLANT PATHOGENS BY SEROLOGICAL METHODS 2.pdfsunilsuriya1
Detection of bacterial plant pathogens by serological methods involves the use of specific antibodies to identify and quantify the presence of harmful bacteria in plants. This approach is based on the principle of antigen-antibody interactions, where antibodies bind to specific antigens on the surface of the target bacteria.
Here's a short description of the process:
1. **Antibody Production**: Specific antibodies are raised against the bacterial antigens of interest. These antibodies can be generated in animals, such as rabbits or mice, through immunization with the target bacterial cells or purified antigens.
2. **Sample Collection**: Plant samples suspected of being infected with the target bacteria are collected from the field. These samples could include leaves, stems, roots, or fruits.
3. **Sample Preparation**: The collected plant samples are processed to extract bacterial antigens. This may involve grinding the plant tissue and isolating the bacterial cells or proteins.
4. **Serological Assay**: The extracted antigens are then applied to a solid phase, such as an enzyme-linked immunosorbent assay (ELISA) plate. The plate is coated with the extracted antigens, allowing them to immobilize on the surface.
5. **Antibody Binding**: The specific antibodies generated earlier are added to the plate. If the target bacteria are present in the sample, the antibodies will bind to the bacterial antigens, forming antigen-antibody complexes.
6. **Detection**: A secondary antibody, often labeled with an enzyme or fluorescent molecule, is then added. This secondary antibody binds to the primary antibodies, amplifying the signal.
7. **Signal Development**: In an ELISA, for example, an enzyme substrate is added, which, upon reaction with the enzyme on the secondary antibody, produces a detectable color change. In fluorescent assays, the signal is detected using a fluorescence microscope or plate reader.
8. **Quantification**: The intensity of the color change or fluorescence is proportional to the amount of target bacteria present in the sample. This allows for the quantification of the bacterial pathogen's concentration in the plant sample.
9. **Interpretation**: Results are compared to standards or controls to determine the presence and concentration of the bacterial pathogen. Positive samples show a visible signal, while negative samples do not.
**Advantages of Serological Methods:**
- High specificity, as antibodies are designed to target specific bacterial antigens.
- Sensitivity to detect even low concentrations of the pathogen.
- Relatively rapid results compared to traditional culture-based methods.
- Applicability to a wide range of plant samples and bacterial pathogens.
**Limitations:**
- Requires specific antibodies for each target pathogen.
- Cross-reactivity with related bacterial species can occur.
- Proper sample handling and processing are crucial to avoid false positives.
1. PANORAIA KYRIAZOPOULOU
E R A S M U S + P L A C E M E N T 2 0 1 5 - 2 0 1 6
Analysis of 2 unique Greek
MHC haplotypes
2. What is the MHC?
The MHC – Major Histocompatibility Complex is a
genetic region comprised of tightly linked genes.
It has been referred to as ovine leukocyte antigen
(OLA) or sheep lymphocyte antigen and now
following the nomenclature system for the MHC of
vertebrates (Klein et al., 1990), it is designated as
‘Ovar-Mhc’ (‘Ovar’ representing Ovis aries).
3. What is the purpose of the MHC?
Purpose:
Encoding molecules playing a central role in
immunological response.
Codes for specialised antigen-presenting receptor
glycoproteins, known as histocompatibility
molecules or MHC molecules.
These molecules bind processed peptide antigens
and present them to T lymphocytes triggering
immune responses.
Communication between cells during immune
response.
4. Why do we study the MHC?
• Alleles of different MHC genes have been found to
contribute in disease resistance/susceptibility.
• The MHC alleles are markers of biodiversity.
• Selection for productivity traits/ disease research.
• Mate choice for many vertebrates through olfactory
cues.
6. MHC Genes
CLASS I GENES
Classical and non-classical genes.
Present peptides to CD8+ cytotoxic T cells.
Interact with natural killer (NK) cells to prevent NK-
mediated cell lysis (Reyburn et al., 1997).
Controversy over the number of classical class I loci - at
least four distinct polymorphic loci have been identified
(Miltiadou et al., 2005).
7. MHC Genes
CLASS II GENES
Class II genes have antigen peptide presenting role to the
TCR on CD4+ helper T cells.
In the HLA complex, these include five sets of the
classical genes DP, DM, DO, DQ, and DR and non-
classical genes such as LMP, TAP and TAPBP.
Not all sets contain genes for both chains, although some
contain many pseudogenes (Tizard, 2004).
8. MHC Genes
Ovar-DR genes
DR genes highly polymorphic
molecules encoded by these genes are expressed in higher
levels.
The DR heterodimer consists of an α-chain and a β-chain,
encoded by DRA and DRB genes.
Class II region of the MHC of sheep (OLA).
DRA genes
DRA gene is considered to be almost monomorphic.
A significantly divergent DRB1 allele (Ovar-DRB1*0901), was
linked to polymorphism at the DRA locus in domestic sheep
(Ballingall et al., 2010).
9. MHC Genes
Ovar-DR genes
DRB genes
DRB locus is the most polymorphic among the MHC
genes.
Ovar-DRB genes exist in multiple copies, some
functional and others non-functional.
Four Ovar-DRB loci have been described (Scott et al.
1991b).
Majority of nucleotide polymorphism at Class II loci
locates in the second exon and adjoining intron 2.
10. MHC Genes
Ovar-DQ genes
DQA genes
Two loci, DQA1 and DQA2, exon 2
A number of haplotypes the DQA1 gene appears absent
(DQA1 null; Fabb et al. 1993) and is replaced by a second
locus more closely related to DQA2 (DQA2-like; Hickford
et al. 2004)
DQA2-like allelic lineage appears functional (Ballingall
et al., 2015).
11. MHC Genes
Ovar-DQ genes
DQB genes
DQB1 and DQB2- exon 2 (Wright and Ballingall, 1994).
Difficulty in assigning sequences to separate loci because
of high similarity between the two DQB genes.
12. Purpose of study
Characterising the polymorphism in the Ovar-MHC
region in a rare Greek sheep breed ~Argos.
Genes studied: DRB1, DRA, DQA and DQB (partial as
well as full-length allelic sequences).
13. Materials and methods
Argos breed:
Originally from Asia Minor.
Single pure flock in Messinia.
Six flocks near Argos with over 50% purity.
Isolated pure specimens in mixed flocks.
14. Materials and methods
Breed details:
Officially recognised breed
Area of distribution: Messinia, Peloponnese
Population size: 100 animals
Risk status: endangered
Colour: white with black head
Fat tailed
Weight ram: 70 kg; ewe: 59 kg
Height ram: 85 cm; ewe: 70 cm
Use: milk, meat
Productivity milkyield: 140-160kg; littersize: 1.5-1.8
15. Materials and methods
At the start of this study, blood was taken from the 2
Argos sheep.
Genomic DNA was prepared.
RNA was extracted and brought to the Moredun
Research Institute.
16. Materials and methods
DRB1 genotyping:
Primers used for sequence based genotyping were 455/329
(Ballingall and Tassi, 2010, and unpublished data).
PCR cycling profile:
1 cycle 5 minutes, 94o C,
35 cycles of 30 s at 94°C,
30 s at 60°C,
30 s at 72°C
followed by 4 min at 72°C.
17. Materials and methods
DRB1 genotyping:
For the mother (Animal 251 or Argos 28) the 3-prime end
was amplified by designing a forward allele-specific
primer 462 (Unpublished data).
Nested approach :primers 455 and 201
annealing temperature at 55°C for 30 cycles
then 2ul PCR product added into the second round with the
new primer and 201
annealing temperature of 60 °C for 35 cycles.
18. Materials and methods
DRB1 Full-length genotyping:
primers 204/207 and 205/207 (Ballingall et al., 2008)
internal primers 222/223
control primers 414/415
PCR cycling profile:
1 cycle 5 minutes, 94o C,
55 cycles of 1min at 94°C,
1 min at 60°C,
1 Min at 72°C
followed by 4 min at 72°C
19. Materials and methods
DQA/DQB genotyping
Primers:
DQA1: 314/315 DQA2: 316/317
DQA2-like: 316/317
DQB1: 363n/ 365n
DQB2:363n/406
PCR cycling profile:
1 cycle 5 minutes, 94o C,
35 cycles of 30 s at 94°C,
30 s at 58°C,
30 s at 72°C
followed by 4 min at 72°C.
DQA/DQB full-length
genotyping:
Primers:
DQA1/2: 283/241
244/241
347/349
DQB1/2: 245/247
246/248
PCR cycling profile:
1 cycle 3 minutes, 94o C,
35 cycles of 30 s at 94°C,
30 s at 55°C,
30 s at 72°C
followed by 3 min at 72°C.
20. Materials and methods
Class I genotyping:
Primers: 415/409
PCR cycling profile:
1 cycle 5 minutes, 94o C,
30 cycles of 30 s at 94°C,
30 s at 55°C,
30 s at 72°C
followed by 4 min at 72°C.
Class I full-length
genotyping:
Primers: 417/411 417/412
410/412 413/411 413/412
PCR cycling profile:
1 cycle 5 minutes, 94o C,
30 cycles of 30 s at 94°C,
30 s at 55°C,
30 s at 72°C
followed by 4 min at 72°C.
21. Materials and methods
Efficiency of PCR was checked on a 1% agarose .
Remaining PCR product purified.
Bidirectional sequencing.
After receiving the first results the selected PCR
fragment was cloned, screened and digested with
RSAI and sent for sequencing.
22. Materials and methods
Patterns when cut with RSAI:
DQB patternsDQA patterns
Class I patterns
23. Materials and methods
Levels of hybridisation were very high for the Class I
using a PCR profile of 30 cycles.
New cDNA was prepared and PCR using primers
416/409 was repeated.
The samples were removed after 12, 14, 16, 18, 20,
22, 25 and 28 cycles and ran on agarose gels 1%.
27. Materials and methods
The gel fragment for 16, 18 and 20 cycles was cut out
and cleaned up.
PCR screening showed that the transformation
worked effectively and samples from colonies
selected were sent for sequencing.
The results received after this procedure showed
significantly lower levels of hybridisation.
29. Results
New DQA1 and DQA2 alleles found.
New DQB1 and 2 DQB2 alleles found.
5 new classical Class I alleles found.
2 new non-classical Class I alleles found.
High levels of diversity
30. Results
Optimal PCR cycles for lower levels of hybridisation
in Class I : 16-20
Hybrids at 30 cycles: 11/30 and PCR errors
Hybrids at 16-20 cycles: 1/40, few PCR errors
This may mean some alleles that appear in low
frequencies might be missed.
MHC molecules act by binding peptide fragments of antigens that have been processed in specialized antigen-presenting cells. Clonally determined antigen receptors on T cells then recognize and bind to specific peptide-MHC complexes, setting into motion the appropriate immune response. Segments of MHC molecules show sequence homologies with immunoglobulins, T cell antigen receptors, and T cell interaction molecules such as CD4 and CD8, which suggests that all these molecules share a common evolutionary ancestry.
(intracellular proteins and parasites)
(mainly derived from extracellular proteins and parasites)
genotyping studies usually focus on the DRB1 gene to associate MHC diversity with resistance or susceptibility to disease, as the DRB1 exon 2 encodes the b1 domain, which constitutes part of the PBR of the DR molecules which in turn are in close contact with the peptides presented in the PBR or the TCR
. Previous analyses described the duplication of the DQA and DQB genes within the MHC of sheep (Scott et al. 1987) with the majority of haplotypes having both DQA1 andDQA2 loci (Hickford et al. 2007). However, in
The efficiency of the PCR was checked by analyzing 10 µl of the PCR product on a 1% agarose gel and the remaining PCR product was then purified to remove excess nucleotides and primers, and the samples were sent for bidirectional sequencing
using the Wizard® SV Gel and PCR Clean-Up System by Promega and ligation and transformation followed. In order to achieve the formation of clones higher concentration was spread on agarose plates (50 μl, 200μl and 250μl).
making it easier to distinguish new alleles avoiding false interpretations of the sequencing results.