This study aimed to express and purify the APOBEC3H (A3H) haplotypes II and VII, which are known to be active against HIV-1. A3H was expressed in insect Sf9 and S2 cells, and purified using GST affinity chromatography. Contaminating nucleic acids were removed through high salt washes, nuclease treatment, and polyethyleneimine precipitation. While A3H could be expressed in both cell lines, higher yields were obtained in Sf9 cells. Further work is needed to stabilize A3H after nucleic acid removal for downstream structural and functional studies.
Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency
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.
Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency
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.
Atherosclerosis: New Insights on Foamy Macrophage Induction and PersistenceDr. Marian Laderoute
It is commonly assumed high cholesterol contributes to atherosclerosis which initiates with foamy macrophages. Here it is suggested the induction of foam in human macrophages relates instead to the induction of a newly described foamy virus, human endogenous retrovirus K102 (HERV-K102) in response to high cortisol and/or viruses, other intracellular pathogens, or tumors. HERV-K102 is a newly described human host protection mechanism and the particles are replication competent in vitro and in vivo. In healthy or younger adults, the foamy macrophages lyse on day 7 and release the protective HERV-K102 particles. When immunosenescence is evident (associated with low DHEA and high cortisol) the lysis and release of the HERV-K102 particles is blocked by alpha-fetoprotein (AFP). The dysfunctional and partly activated macrophages promote immunosenescence including atherosclerosis. See Laderoute MP, Discovery Medicine article published December 2015 for more details.
In silico functional assignment to Rv3430c, a Mycobacterium tuberculosis gene...Rajni Chauhan
I analysed a previously published transcriptomics study and found that Mtb gene
Rv3430c, of unknown function, was upregulated 5-fold and 3-fold, when grown on
blood from HIV positive and negative people, respectively. In the present study,
Rv3430c was subjected to various bioinformatic analysis based on its primary
amino acid sequence. Further, a 3-D computational model of Rv3430c was built
and the structure was analysed to predict its function. The structural analysis
showed that its N-terminal has helix-turn-helix structural motif, the central core has
the conserved catalytic integrase core domain which contains a catalytic triad of
three highly conserved residues, DDE motif, and the C-terminal contains an SH3-
Technical Expertise:
Bioinformatics and statistical tools:
Biochemical Techniques:
.
Molecular biology techniques:
Microbiological and
Immunological techniques:
Biophysical Techniques:
Software Skills:
Presentations:
Seminar on topic “Synthetic Biology and Using Synthetic RNAs as Scaffold to co-localize multiple protein and Regulators to control the gene expression.” at Panjab University Chandigarh, Department of Biochemistry on March 2015.
Journal club on topic “Potential Pharmacological Chaperones Targeting Cancer –Associated MCL-1 and Parkinson Disease-Associated Alfa-Synuclein.” at Panjab University Chandigarh, Department of Biochemistry on September 2014.
Certifications:
For Comprehensive VANTAGE Course in English speaking / Personality Development
For participation in “UNESCO INTERNATIONAL ASSAY CONTEST 2015”
UniProt, SVMProt, EcoCyc, Pfam, SCOP, Phobius, BLAST. FASTA, ClustalW, LOMETS, VADAR, Dali Server, ICM, PDB, Multiple Sequence alignment and SPSS.
Isolation and purification of proteins, TLC, Ion exchange, Gel exclusion chromatography, SDS and native PAGE and Enzyme activity assay, Kidney and Liver function tests, Enzyme profile
during myocardial infarction, Western Blotting.
Recombinant DNA technology, Methylation Specific PCR, Isolation of DNA, DNA Quantification, primer design, Cloning
and expression of genetic constructs, etc.
Bacterial culture, bacterial growth curve, antibiotic sensitivity tests, microscopy, ELISA, Immunohistochemistry assay, Flow Cytometer, Western blotting, Immunoblot, Immunodiffusion, cell culturing techniques etc
UV-visible spectroscopy, GM counters radioactive decay analysis, Fluorescent microscopy, etc.
Ubuntu, Windows, Photoshop, Sigma Plot, Power point, Excel
fold motif. The Rv3430c model could be structurally super-imposed onto multiple integrases having no significant sequence identity with an RMSD of ≤ 2.8 Å. The analysis of DNA binding pocket of Rv3430c and its comparison with 3-D structures of other integrases showed that despite no sequence identity with other integrases, the DNA binding pocket and amino acids in the active site are conserved. This work shows that Rv3430c is likely to be an integrase.
Blood leukocytes have a vital role in easing general inflammation through acute pancreatitis. Irrespective of topical growths in the considerate of the intricate pathogenesis of pancreatitis, the existence of the disease remnants sub-optimal. The present work attempts to detect the gene expression level of Fas Ligand (FasL) cDNA in four blood samples. The blood samples consist of different age groups (7 years, 18 years, 25 years, and 50 years), whose expression for the gene FasL was studied by real time PCR. From the study, it was observed, in the sample no (5) of 50 years age the expression is highest followed by sample no (1) of 25 years > sample no (4) of 18 years > sample no (3) of 7 years. The FasL gene is over expressed in the sample no (5) and under expressed in the sample no (1), (4), and (3).
Key-words: FasL, cDNA, Cycle threshold, Apoptosis, Lymphocytes, Severe acute pancreatitis, Immunosuppression
Atherosclerosis: New Insights on Foamy Macrophage Induction and PersistenceDr. Marian Laderoute
It is commonly assumed high cholesterol contributes to atherosclerosis which initiates with foamy macrophages. Here it is suggested the induction of foam in human macrophages relates instead to the induction of a newly described foamy virus, human endogenous retrovirus K102 (HERV-K102) in response to high cortisol and/or viruses, other intracellular pathogens, or tumors. HERV-K102 is a newly described human host protection mechanism and the particles are replication competent in vitro and in vivo. In healthy or younger adults, the foamy macrophages lyse on day 7 and release the protective HERV-K102 particles. When immunosenescence is evident (associated with low DHEA and high cortisol) the lysis and release of the HERV-K102 particles is blocked by alpha-fetoprotein (AFP). The dysfunctional and partly activated macrophages promote immunosenescence including atherosclerosis. See Laderoute MP, Discovery Medicine article published December 2015 for more details.
In silico functional assignment to Rv3430c, a Mycobacterium tuberculosis gene...Rajni Chauhan
I analysed a previously published transcriptomics study and found that Mtb gene
Rv3430c, of unknown function, was upregulated 5-fold and 3-fold, when grown on
blood from HIV positive and negative people, respectively. In the present study,
Rv3430c was subjected to various bioinformatic analysis based on its primary
amino acid sequence. Further, a 3-D computational model of Rv3430c was built
and the structure was analysed to predict its function. The structural analysis
showed that its N-terminal has helix-turn-helix structural motif, the central core has
the conserved catalytic integrase core domain which contains a catalytic triad of
three highly conserved residues, DDE motif, and the C-terminal contains an SH3-
Technical Expertise:
Bioinformatics and statistical tools:
Biochemical Techniques:
.
Molecular biology techniques:
Microbiological and
Immunological techniques:
Biophysical Techniques:
Software Skills:
Presentations:
Seminar on topic “Synthetic Biology and Using Synthetic RNAs as Scaffold to co-localize multiple protein and Regulators to control the gene expression.” at Panjab University Chandigarh, Department of Biochemistry on March 2015.
Journal club on topic “Potential Pharmacological Chaperones Targeting Cancer –Associated MCL-1 and Parkinson Disease-Associated Alfa-Synuclein.” at Panjab University Chandigarh, Department of Biochemistry on September 2014.
Certifications:
For Comprehensive VANTAGE Course in English speaking / Personality Development
For participation in “UNESCO INTERNATIONAL ASSAY CONTEST 2015”
UniProt, SVMProt, EcoCyc, Pfam, SCOP, Phobius, BLAST. FASTA, ClustalW, LOMETS, VADAR, Dali Server, ICM, PDB, Multiple Sequence alignment and SPSS.
Isolation and purification of proteins, TLC, Ion exchange, Gel exclusion chromatography, SDS and native PAGE and Enzyme activity assay, Kidney and Liver function tests, Enzyme profile
during myocardial infarction, Western Blotting.
Recombinant DNA technology, Methylation Specific PCR, Isolation of DNA, DNA Quantification, primer design, Cloning
and expression of genetic constructs, etc.
Bacterial culture, bacterial growth curve, antibiotic sensitivity tests, microscopy, ELISA, Immunohistochemistry assay, Flow Cytometer, Western blotting, Immunoblot, Immunodiffusion, cell culturing techniques etc
UV-visible spectroscopy, GM counters radioactive decay analysis, Fluorescent microscopy, etc.
Ubuntu, Windows, Photoshop, Sigma Plot, Power point, Excel
fold motif. The Rv3430c model could be structurally super-imposed onto multiple integrases having no significant sequence identity with an RMSD of ≤ 2.8 Å. The analysis of DNA binding pocket of Rv3430c and its comparison with 3-D structures of other integrases showed that despite no sequence identity with other integrases, the DNA binding pocket and amino acids in the active site are conserved. This work shows that Rv3430c is likely to be an integrase.
Blood leukocytes have a vital role in easing general inflammation through acute pancreatitis. Irrespective of topical growths in the considerate of the intricate pathogenesis of pancreatitis, the existence of the disease remnants sub-optimal. The present work attempts to detect the gene expression level of Fas Ligand (FasL) cDNA in four blood samples. The blood samples consist of different age groups (7 years, 18 years, 25 years, and 50 years), whose expression for the gene FasL was studied by real time PCR. From the study, it was observed, in the sample no (5) of 50 years age the expression is highest followed by sample no (1) of 25 years > sample no (4) of 18 years > sample no (3) of 7 years. The FasL gene is over expressed in the sample no (5) and under expressed in the sample no (1), (4), and (3).
Key-words: FasL, cDNA, Cycle threshold, Apoptosis, Lymphocytes, Severe acute pancreatitis, Immunosuppression
A Power Point Presentation on some Riddles to train the mind to think divergently. Done by Bro. Oh Teik Bin, Lower Perak Buddhist Association, Teluk Intan, Malaysia.
A Power Point Presentation for children, students and the young to stimulate their logical, creative and divergent thinking...to think out of the box. Please download for some animated images.
In vitro transcription and transfection of HCV genomic repliconBinodGupta27
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
In vitro transcription and transfection of HCV genomic repliconBinodGupta27
ABSTRACT:
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
ABSTRACT- Multiple Drug resistance (MDR) tuberculosis timely diagnose is of utmost clinical relevance and needs to be diagnose at initial stages for the proper treatment. The current study was done to detect the several genes for MDR tuberculosis (TB) in clinical isolates by molecular tools. 60 clinical isolates were collected and subjected for AFB smear preparation, Nested PCR (IS6110) for mycobacterium tuberculosis complex detection and MDR TB PCR targeting rpoB, kat G, mab A promoter. 12 came positive for AFB smears, out of which 08 were pulmonary and 04 were extra pulmonary. Nested PCR targeting IS6110 gene was amplified at 123 base pairs with 340 base pairs as IC (internal control) was seen in 25 cases which include 19 pulmonary and 6 extra pulmonary. The Positive TB PCR specimens were subjected for MDRTB PCR Only 06 cases yielded, an amplicon of 315 bp confirming the rpoB gene resistance for resistance for rifampcin drug. In any of the 06 positives none of the other resistance gene other than rpoB was amplified. Targeting multiple genes at once, additional information will be gained from a single test run that otherwise would require several times the reagents and more time to perform. Current study signifies the usage of quick, cost effective, DNA sequences based method for MDR TB detection where disease will be diagnosed earlier and hence treatment would be started at an early stage.
Keywords: Multiple drug resistance, amplicon, Polymerase chain reaction, Nested PCR, Rifampicin.
AMPLIFICATION OF rpoB, kat G & mab A (fab G1)- inh A PROMOTOR DNA SEQUENCES B...
POSTER-MARIA KLARIZZA PANALIGAN
1. A3H II EXPRESSED IN SF9 CELLS
GST affinity
purification
Optimization of the expression and purification of APOBEC3H
Maria Klarizza Panaligan, Karen Siu, Jeffrey E. Lee
Department of Laboratory Medicine & Pathobiology, University of Toronto
Introduction
A3H proteins are restriction factors that contribute to the
innate immune response against retroviruses, such as the
human immunodeficiency virus type 1 (HIV-1).
FUNCTION
• DNA cytidine
deaminase
that generate
C-to-U mutations
on the viral
(–) ssDNA strand
Rationale
The structure of A3H has not yet been solved, and little is known
of its haplotypes (II and VII) that are active against HIV-1. In our
study, we focused on isolating sufficient amounts of stable A3H
that will be useful for future downstream experiments.
Methods & Results
PROTEIN EXPRESSION AND PURIFICATION
• A3H forms inclusion bodies when expressed from E.coli
• Recombinant protein expression in Sf9 and S2 insect cells
• After PEI precipitation,
nanodrop 260/280 readings
dropped from ~1.51 to ~0.5
Discussion
Target Cell
C
A3H
C U
A
reverse
transcripted
(-) strand ssDNA
C-to-U deamination
by A3H
Deaminated
(-) ssDNA
(+) ssDNA
(G-to-A)
ANTAGONIST
• Antagonized by an HIV-1 protein called Vif
A3H +
Vif
• A3H can be expressed in S2 cells, but in lower
amounts compared to Sf9 cells
• PEI precipitation is an efficient method for removing
nucleic acid contaminants from A3H
• A3H becomes “sticky” and is difficult to elute from
the resin after removal of nucleic acids
Future Directions
• A260/280 readings indicate
nucleic acid contamination
in purified A3H II and VII
from both Sf9 and S2 cells.
• Needed to remove nucleic
acids in order to study A3H
• Intracellular expression of
A3H confirmed in S2 cells
(+) (+) media
A3H II and VII
from S2 cells
western blot
S2 cell
media
western blot
hap
II
hap
VII
ANTIVIRAL ACTIVITY
infected
cell
healthy
cell
• Hypermutates viral DNA, resulting in a genome that can no
longer be used for viral replication in the next host cell.
unsuccesful
infection
Human cells have developed multiple mechanisms to inhibit viral replication, and viruses
in turn have evolved diverse strategies to antagonize these efforts. Apolipoprotein B
mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are a key factor
in an intracellular defense strategy for inhibition of replication of HIV. While seven
haplotypes of APOBEC3H are present in humans, only haplotypes II, V and VII are active
against HIV-1. These haplotypes are packaged into newly assembled virions and induce
C-to-U mutations on the viral minus-strand DNA, which results in a hypermutated
genome that would be unable to infect the next target cell. HIV-1 disrupts this host
defense mechanism by encoding a viral infectivity factor (Vif) that effectively targets
APOBEC3 proteins for E3 ubiquitin ligase-mediated proteasomal degradation. Stable
APOBEC3H haplotypes have been shown to show some catalytic activity even in the
presence of Vif. The study of the HIV-1 Vif/APOBEC3 axis represents an attractive target
for HIV/AIDS research. Biochemical and high-resolution structural analysis of the
interplay between HIV-1 Vif, APOBEC3s and other cellular factors provide an integrated
approach to understand their function and to provide a master blueprint to design new
classes of HIV drugs that unleash the potent anti-viral activity of host innate immune
factors for AIDS prevention/therapy. Here, we present our preliminary work towards
expressing and purifying various full-length APOBEC3H haplotypes for future functional
and structural studies.
HIV-1 Vif
close-up
diagram
Abstract
Method A260/280
for A3H II
High salt washes
Benzonase + RNase A
treatment
PEI precipitation 0.5
1.52
1M NaCl
1M LiCl
2M NaBr
1.47
1.59
1.46
haplotype
# 15 18 105 121 178
I
II
III
IV
V
VI
VII
N R G K E
N R R D D
R R D D
L R D D
N R R D E
L G K D
N R R K E
anti-
HIV
activity
SF9/BACULOVIRUS DROSOPHILA S2
Method used virus infection transfection
Viability declines after
infection
constantly
high
Cell line
GST-affinity purification
GST
GSH
A3H
GST
A3H
GSH
GSHGST
+
thrombin
A3H
• A3H II and VII expressed in and purified from Sf9 cells
GST tag (~26kDa)
A3H+GST tag
(~48kDa)
REMOVING CONTAMINATING NUCLEIC ACID FROM A3HII
4.) Nucleic acid content was monitored through 260/280
NanoDrop 2000 readings
PEI
A3H
A3H
A3H
PEI
PEI
A3H
A3H
A3H
PEI
A3H
A3H
A3H
PEI
A3H A3H
A3H
+PEI
+non-saturating
ammonium
sulfate
rock 4C
for
20min
and spin
resuspend pellet
for dialysis and
GST
PURIFICATION
PROTOCOL
PEI
PEI
PEI
+ saturated
ammonium
sulfate
and spin
PEI
PEI
1.) High salt washes during GST-affinity purification.
2.) Benzonase and RNase A treatment
3.) PEI precipitation
A3H+
GST tag
A3H
PEI
1A1 1A2 1A3
Ion Exchange Chromatography
GST affinity purification
coomasie gel
A3HII
lysate
A3HII
beads
A3HVII
lysate
A3HVII
beads
MW
(kDa)
MW
(kDa)
1A1 1A2 1A3
1A1 1A2 1A3
A3H VII
Mono S run
A3H II
Mono S run
A3H II
size column run
Size Exclusion Chromatography
1A6-1A10
MW 1A61A71A81A91A10
1A1-1A8
1A1-
1A8
MW
(kDa)
A3H VII
size column run
1A1 1A2 1A3
BIOCHEMICAL PROFILE
• 7 haplotypes: I II III IV V VI VII
Ub
Ub
proteasomal
degradation
degraded
A3H
A3H
Ub
Vif
EloB
EloC
CUL5
RBX2 E2 Ub ACKNOWLEDGEMENTS:
Prof. Linda Chelico,
University of Saskatchewan
coomasie
gel
coomasie
gel
coomasie
gel coomasie
gel
thrombin-
cleaved
A3H
1) Search for additives or detergents that help stabilize A3H
after nucleic acid removal.
2) If insect cell expression remains unpromising, move on
to expressing in mammalian cells
2) Study the binding of A3H with ssDNA substrate
3) Study the binding of A3H with its viral antagonist, Vif
4) Understand the structure of A3H through X-ray
crystallography and other
biophysical methods
coomasie
gel
45
25
35
20
45
25
35
20
45
25
35
20
45
25
35
20
MW
(kDa)
45
25
35
20
45
25
35
20
45
25
35
20
45
25
35
20
(kDa)
MW
(kDa)
