This document summarizes research on the impact of genetic variation in the HIV-1 long terminal repeat (LTR) and trans-activator of transcription (Tat) protein on HIV-1 transcription and disease progression. The research examines samples from an HIV/AIDS genetic analysis cohort to identify single nucleotide polymorphisms (SNPs) in the LTR associated with CD4 count, viral load, and disease severity over time. Functional analysis of Tat proteins from patient samples shows variation at amino acid position 100 that correlates with neurocognitive impairment.
The TX-2B/RX-2B is a pair of CMOS LSIs designed for remote controlled car applications. It has five control keys for controlling motions like forward, backward, right, left, and turbo functions. It features a wide operating voltage range, low stand-by current, and auto-power-off function. Few external components are needed. The TX-2B transmits encoded signals corresponding to the key pressed and the RX-2B receives and decodes the signals to control the car motions.
This document summarizes the features and specifications of the ATmega16 microcontroller. It has a low-power 8-bit AVR processor with 16K bytes of flash memory, 512 bytes of EEPROM, and 1K byte of SRAM. It includes timers, PWM channels, ADC, serial interfaces, and I/O pins. The microcontroller supports in-system programming of its flash memory and operates between 0-16MHz with voltages from 2.7-5.5V.
The AT89C51 is a low-power, high-performance 8-bit microcontroller with 4K bytes of flash memory. It has 32 I/O lines, two 16-bit timer counters, serial communication capabilities, and on-chip programming. The device operates between 0 and 24 MHz and has various power saving modes like idle and power down to reduce power consumption. It is compatible with the MCS-51 instruction set and provides a cost-effective solution for embedded applications.
This document contains code for using a Proteus simulation with a PIC microcontroller to blink an LED using different methods. It defines ports and pins as inputs and outputs, and uses pause commands to turn an LED on and off at set time intervals. It also includes code for initializing an LCD display using specific port pins and bits.
The TB62206FG is a bipolar stepping motor driver IC that uses BiCD process technology. It can drive a 2-phase stepping motor with an output withstand voltage of 40V and maximum current of 1.8A per phase. The IC features internal PWM current control, 2-phase or 1-2 phase excitation, monolithic BiCD construction, and on-chip protection circuits. It is packaged in an HSOP20-P-450-1.00 package.
1. The document discusses interrupts in the 8085 microprocessor. It lists the 5 hardware interrupts - INTR, RST 5.5, RST 6.5, RST 7.5, and TRAP - and their priorities, with TRAP being the highest. It distinguishes between vectored interrupts, which provide the interrupt address, and non-vectored interrupts, where the address must be supplied.
2. An example assembly language program is provided to create a time delay of 1.8 seconds using nested loops and decrement instructions. Interrupt basics are reviewed, such as the differences between maskable and non-maskable interrupts.
This document provides standard operating procedures for safely operating the 6.6kV switchboard at NMDC. It outlines safety precautions, roles and responsibilities, and step-by-step procedures for switching various feeders on and off, including incomer, bus coupler, and outgoing feeders. The procedures describe checking equipment status, obtaining clearances, verifying voltages and breaker positions before executing switch operations either locally or remotely via SCADA. Emergency conditions and general instructions are also covered.
The TX-2B/RX-2B is a pair of CMOS LSIs designed for remote controlled car applications. It has five control keys for controlling motions like forward, backward, right, left, and turbo functions. It features a wide operating voltage range, low stand-by current, and auto-power-off function. Few external components are needed. The TX-2B transmits encoded signals corresponding to the key pressed and the RX-2B receives and decodes the signals to control the car motions.
This document summarizes the features and specifications of the ATmega16 microcontroller. It has a low-power 8-bit AVR processor with 16K bytes of flash memory, 512 bytes of EEPROM, and 1K byte of SRAM. It includes timers, PWM channels, ADC, serial interfaces, and I/O pins. The microcontroller supports in-system programming of its flash memory and operates between 0-16MHz with voltages from 2.7-5.5V.
The AT89C51 is a low-power, high-performance 8-bit microcontroller with 4K bytes of flash memory. It has 32 I/O lines, two 16-bit timer counters, serial communication capabilities, and on-chip programming. The device operates between 0 and 24 MHz and has various power saving modes like idle and power down to reduce power consumption. It is compatible with the MCS-51 instruction set and provides a cost-effective solution for embedded applications.
This document contains code for using a Proteus simulation with a PIC microcontroller to blink an LED using different methods. It defines ports and pins as inputs and outputs, and uses pause commands to turn an LED on and off at set time intervals. It also includes code for initializing an LCD display using specific port pins and bits.
The TB62206FG is a bipolar stepping motor driver IC that uses BiCD process technology. It can drive a 2-phase stepping motor with an output withstand voltage of 40V and maximum current of 1.8A per phase. The IC features internal PWM current control, 2-phase or 1-2 phase excitation, monolithic BiCD construction, and on-chip protection circuits. It is packaged in an HSOP20-P-450-1.00 package.
1. The document discusses interrupts in the 8085 microprocessor. It lists the 5 hardware interrupts - INTR, RST 5.5, RST 6.5, RST 7.5, and TRAP - and their priorities, with TRAP being the highest. It distinguishes between vectored interrupts, which provide the interrupt address, and non-vectored interrupts, where the address must be supplied.
2. An example assembly language program is provided to create a time delay of 1.8 seconds using nested loops and decrement instructions. Interrupt basics are reviewed, such as the differences between maskable and non-maskable interrupts.
This document provides standard operating procedures for safely operating the 6.6kV switchboard at NMDC. It outlines safety precautions, roles and responsibilities, and step-by-step procedures for switching various feeders on and off, including incomer, bus coupler, and outgoing feeders. The procedures describe checking equipment status, obtaining clearances, verifying voltages and breaker positions before executing switch operations either locally or remotely via SCADA. Emergency conditions and general instructions are also covered.
The document provides information about the 74HC/HCT4020 integrated circuit, which is a 14-stage binary ripple counter. It has 12 parallel outputs, a clock input, and an overriding asynchronous master reset input. The counter advances on the falling edge of the clock input and the master reset input asynchronously clears all counter stages and forces the outputs low. The document includes specifications, pin descriptions, logic diagrams, timing diagrams, and package information.
This document provides standard operating procedures for switching various types of 6.6kV feeders, including incomer, bus coupler, and outgoing feeders, on and off. It outlines the roles and responsibilities, safety precautions, requirements before starting work, and step-by-step processes for operating each type of feeder in various conditions. The procedures describe coordinating with control room operators, checking equipment status, obtaining clearances, and documenting the work in a logbook.
These documents describe several high-performance PAL circuits from Texas Instruments. The circuits provide functionality equivalent to existing PAL devices but with higher maximum frequencies of up to 62.5MHz. They integrate Advanced Low-Power Schottky technology with titanium-tungsten fuses. The devices have preload capability on output registers to simplify testing. They are available in plastic and ceramic packages over commercial and extended temperature ranges.
Embedded Systems Training & Live Projects @Technogroovy Systems India Pvt Ltd Technogroovy India
ย
This document discusses numerical bases used in programming such as hexadecimal, binary, and BCD. It provides examples of converting between decimal, binary, hexadecimal, and BCD representations of numbers. It also discusses registers, memory mapping, addressing modes, instructions, and other concepts related to the 8051 microcontroller.
This document describes an 8-bit microcontroller with features including a powerful instruction set, programmable flash memory and EEPROM, USB and JTAG interfaces, timers, PWM channels, ADC, and I/O pins. It has 64/128KB of flash memory, 2/4KB of EEPROM, and supports USB 2.0 device functionality and OTG dual-role capability. It is suitable for industrial applications with an operating temperature range of -40ยฐC to +85ยฐC.
1. A hardware reset which initializes all on-chip registers
including the SFRs.
The AT89C51 is a low-power, high-performance 8-bit micro-
controller with 4K bytes of flash memory. It has 32 I/O lines, 2. Return from a CALL instruction. In this case, the pro-
two 16-bit timer/counters, serial communication capabilities, gram counter (PC) and registers will resume their values
and low-power idle and power-down modes. The document prior to entry into power-down.
provides details on the microcontroller's features, pinout, 3. An external interrupt condition that causes a wake
Beyond Degrees - Empowering the Workforce in the Context of Skills-First.pptxEduSkills OECD
ย
Ivรกn Bornacelly, Policy Analyst at the OECD Centre for Skills, OECD, presents at the webinar 'Tackling job market gaps with a skills-first approach' on 12 June 2024
Philippine Edukasyong Pantahanan at Pangkabuhayan (EPP) CurriculumMJDuyan
ย
(๐๐๐ ๐๐๐) (๐๐๐ฌ๐ฌ๐จ๐ง ๐)-๐๐ซ๐๐ฅ๐ข๐ฆ๐ฌ
๐๐ข๐ฌ๐๐ฎ๐ฌ๐ฌ ๐ญ๐ก๐ ๐๐๐ ๐๐ฎ๐ซ๐ซ๐ข๐๐ฎ๐ฅ๐ฎ๐ฆ ๐ข๐ง ๐ญ๐ก๐ ๐๐ก๐ข๐ฅ๐ข๐ฉ๐ฉ๐ข๐ง๐๐ฌ:
- Understand the goals and objectives of the Edukasyong Pantahanan at Pangkabuhayan (EPP) curriculum, recognizing its importance in fostering practical life skills and values among students. Students will also be able to identify the key components and subjects covered, such as agriculture, home economics, industrial arts, and information and communication technology.
๐๐ฑ๐ฉ๐ฅ๐๐ข๐ง ๐ญ๐ก๐ ๐๐๐ญ๐ฎ๐ซ๐ ๐๐ง๐ ๐๐๐จ๐ฉ๐ ๐จ๐ ๐๐ง ๐๐ง๐ญ๐ซ๐๐ฉ๐ซ๐๐ง๐๐ฎ๐ซ:
-Define entrepreneurship, distinguishing it from general business activities by emphasizing its focus on innovation, risk-taking, and value creation. Students will describe the characteristics and traits of successful entrepreneurs, including their roles and responsibilities, and discuss the broader economic and social impacts of entrepreneurial activities on both local and global scales.
Temple of Asclepius in Thrace. Excavation resultsKrassimira Luka
ย
The temple and the sanctuary around were dedicated to Asklepios Zmidrenus. This name has been known since 1875 when an inscription dedicated to him was discovered in Rome. The inscription is dated in 227 AD and was left by soldiers originating from the city of Philippopolis (modern Plovdiv).
This presentation was provided by Rebecca Benner, Ph.D., of the American Society of Anesthesiologists, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
The document provides information about the 74HC/HCT4020 integrated circuit, which is a 14-stage binary ripple counter. It has 12 parallel outputs, a clock input, and an overriding asynchronous master reset input. The counter advances on the falling edge of the clock input and the master reset input asynchronously clears all counter stages and forces the outputs low. The document includes specifications, pin descriptions, logic diagrams, timing diagrams, and package information.
This document provides standard operating procedures for switching various types of 6.6kV feeders, including incomer, bus coupler, and outgoing feeders, on and off. It outlines the roles and responsibilities, safety precautions, requirements before starting work, and step-by-step processes for operating each type of feeder in various conditions. The procedures describe coordinating with control room operators, checking equipment status, obtaining clearances, and documenting the work in a logbook.
These documents describe several high-performance PAL circuits from Texas Instruments. The circuits provide functionality equivalent to existing PAL devices but with higher maximum frequencies of up to 62.5MHz. They integrate Advanced Low-Power Schottky technology with titanium-tungsten fuses. The devices have preload capability on output registers to simplify testing. They are available in plastic and ceramic packages over commercial and extended temperature ranges.
Embedded Systems Training & Live Projects @Technogroovy Systems India Pvt Ltd Technogroovy India
ย
This document discusses numerical bases used in programming such as hexadecimal, binary, and BCD. It provides examples of converting between decimal, binary, hexadecimal, and BCD representations of numbers. It also discusses registers, memory mapping, addressing modes, instructions, and other concepts related to the 8051 microcontroller.
This document describes an 8-bit microcontroller with features including a powerful instruction set, programmable flash memory and EEPROM, USB and JTAG interfaces, timers, PWM channels, ADC, and I/O pins. It has 64/128KB of flash memory, 2/4KB of EEPROM, and supports USB 2.0 device functionality and OTG dual-role capability. It is suitable for industrial applications with an operating temperature range of -40ยฐC to +85ยฐC.
1. A hardware reset which initializes all on-chip registers
including the SFRs.
The AT89C51 is a low-power, high-performance 8-bit micro-
controller with 4K bytes of flash memory. It has 32 I/O lines, 2. Return from a CALL instruction. In this case, the pro-
two 16-bit timer/counters, serial communication capabilities, gram counter (PC) and registers will resume their values
and low-power idle and power-down modes. The document prior to entry into power-down.
provides details on the microcontroller's features, pinout, 3. An external interrupt condition that causes a wake
Beyond Degrees - Empowering the Workforce in the Context of Skills-First.pptxEduSkills OECD
ย
Ivรกn Bornacelly, Policy Analyst at the OECD Centre for Skills, OECD, presents at the webinar 'Tackling job market gaps with a skills-first approach' on 12 June 2024
Philippine Edukasyong Pantahanan at Pangkabuhayan (EPP) CurriculumMJDuyan
ย
(๐๐๐ ๐๐๐) (๐๐๐ฌ๐ฌ๐จ๐ง ๐)-๐๐ซ๐๐ฅ๐ข๐ฆ๐ฌ
๐๐ข๐ฌ๐๐ฎ๐ฌ๐ฌ ๐ญ๐ก๐ ๐๐๐ ๐๐ฎ๐ซ๐ซ๐ข๐๐ฎ๐ฅ๐ฎ๐ฆ ๐ข๐ง ๐ญ๐ก๐ ๐๐ก๐ข๐ฅ๐ข๐ฉ๐ฉ๐ข๐ง๐๐ฌ:
- Understand the goals and objectives of the Edukasyong Pantahanan at Pangkabuhayan (EPP) curriculum, recognizing its importance in fostering practical life skills and values among students. Students will also be able to identify the key components and subjects covered, such as agriculture, home economics, industrial arts, and information and communication technology.
๐๐ฑ๐ฉ๐ฅ๐๐ข๐ง ๐ญ๐ก๐ ๐๐๐ญ๐ฎ๐ซ๐ ๐๐ง๐ ๐๐๐จ๐ฉ๐ ๐จ๐ ๐๐ง ๐๐ง๐ญ๐ซ๐๐ฉ๐ซ๐๐ง๐๐ฎ๐ซ:
-Define entrepreneurship, distinguishing it from general business activities by emphasizing its focus on innovation, risk-taking, and value creation. Students will describe the characteristics and traits of successful entrepreneurs, including their roles and responsibilities, and discuss the broader economic and social impacts of entrepreneurial activities on both local and global scales.
Temple of Asclepius in Thrace. Excavation resultsKrassimira Luka
ย
The temple and the sanctuary around were dedicated to Asklepios Zmidrenus. This name has been known since 1875 when an inscription dedicated to him was discovered in Rome. The inscription is dated in 227 AD and was left by soldiers originating from the city of Philippopolis (modern Plovdiv).
This presentation was provided by Rebecca Benner, Ph.D., of the American Society of Anesthesiologists, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
This document provides an overview of wound healing, its functions, stages, mechanisms, factors affecting it, and complications.
A wound is a break in the integrity of the skin or tissues, which may be associated with disruption of the structure and function.
Healing is the bodyโs response to injury in an attempt to restore normal structure and functions.
Healing can occur in two ways: Regeneration and Repair
There are 4 phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. This document also describes the mechanism of wound healing. Factors that affect healing include infection, uncontrolled diabetes, poor nutrition, age, anemia, the presence of foreign bodies, etc.
Complications of wound healing like infection, hyperpigmentation of scar, contractures, and keloid formation.
How Barcodes Can Be Leveraged Within Odoo 17Celine George
ย
In this presentation, we will explore how barcodes can be leveraged within Odoo 17 to streamline our manufacturing processes. We will cover the configuration steps, how to utilize barcodes in different manufacturing scenarios, and the overall benefits of implementing this technology.
1. Impact of Genetic Variation within the HIV-1 LTR and
Tat on Transcription
David Cunningham
MMS Presentation
Department of Microbiology and Immunology
Center for Molecular Virology and Translational Neuroscience
Drexel University College of Medicine
DREXEL MEDICINE
2. HIV-1 virion structure and genomic organization
Adapted from http://www.stanford.edu/group/virus/retro/2005gongishmail/HIV.html
DREXEL MEDICINE
3. Physical structure of the HIV-1 LTR
500 400 300 200 100 +1 100 200
U3 R U5
AP-1 NFAT-1 USF NF-๏ซB TATA CTF/NFI
NRRE AP-1 AP-1 SP-50 cETS-1 Sp HIP116 AP-1 AP-1
100kD 38kD C/EBP C/EBP E box YY-1 c-fos c-fos
GRE URS LEF-1 LBP-1
ATF/CREB C/EBP TDP-43
MODULATORY REGION ENHANCER REGION CORE REGION
DREXEL MEDICINE
4. Regulation of HIV-1 LTR basal and stimulated transcription
- 405 - 245 + 20 + 165
U3 R U5
Cell activation
TNF-ฮฑ, IL-1ฮฒ, HDAC1
IL-6, IL-7, etcโฆ p50
p50 CTD
TAR NELF TAT
DSIF
Ac Ac p65 Ac
Nuc-0 p50 Nuc-1
TFIID
TATA
C/EBP US2
AP3-L
C/EBP DS3
C/EBP US1
NF-๏ซB
ATF/CREB
AP-1
SP1
Ac Ac HAT
Ac Ac
Modulatory region Core region
Enhancer region
DREXEL MEDICINE
5. Regulation of HIV-1 Tat-mediated LTR activation
- 405 - 245 + 20 + 165
U3 R U5
PCAF
cdk9
CTD
cycT1
TAR
TAT
Ac Ac p65 Ac
Nuc-0 p50 Nuc-1
TFIID
TATA
AP3-L
C/EBP DS3
C/EBP US2
C/EBP US1
NF-๏ซB
ATF/CREB
AP-1
SP1
Ac Ac Ac HAT Ac
Modulatory region Core region
Enhancer region
DREXEL MEDICINE
6. Hypothesis
โข Genetic variation within HIV-1 LTR and Tat co-
select due to selective pressures applied to the
HIV-1 genome during replication, resulting in
altered function of the LTR, Tat, or both.
7. DREXELMED HIV/AIDS Genetic Analysis Cohort in the HAART era
Ficoll-Pacque Qiagen DNEasy
Whole Blood Plus gradient Tissue Kit
Serum & PBMC
separation Isolation of PCR amplify
BSL-3 Facility genomic DNA proviral DNA
Serum and cell banking Separate on
Clinical and virus/host genomic data management agarose gel
HIV-1 Sequence
Database
Sequence PCR product
Analysis sequencing
Gel extraction
pGL3 Basic pCR4-TOPO
Incubate with
Functional PCR amplify/ Taq to add A
analysis clone proviral DNA overhang
DREXEL MEDICINE
8. Summary of DREXELMED HIV/AIDS Genetic Analysis Cohort
ENROLLMENT UPDATE: March 20, 2012
Definitive LTR
Visit1 Number
sequences2
R003 486 400 1On average,
patients are seen
R01 271 195 every six months
R02 185 122
R03 120 72 2Definitive
LTR
sequences as called
R04 81 31
by Brian Moldover,
R05 51 15 Ph.D.
R06 32 4
3R00 has 1481
R07 14
definitive LTR clones
R08 5 from 110 patients
R09 1
TOTALS 1246 839
DREXEL MEDICINE
9. Association analysis of SNPs with regard to CD4 count and log
viral load
Phenotype Position TF site Ref/Mut Mutant Frequency Effect p-value
CD4 244 Pet-1 A/G 56.0% 37.630 0.0233 Effect = change in CD4
293 USF G/ACT 11.5% -54.487 0.0366 count away from the
444 Oct 1 T/C 36.9% -65.098 0.0478 average
Viral Load 108 COUP/AP1 A/CGT 37.3% 153.8% 0.0263
115 unk A/GT 18.9% 61.8% 0.0418
131 unk A/CGT 12.2% 176.1% 0.0409 Effect = % change in VL
168 unk G/ACT 16.2% 56.4% 0.0156 away from the average
385 Sp III (9) G/AC 8.2% 229.6% 0.0242
605 unk C/AT 8.9% 756.8% 0.0308
adjusted for sex, age, race, days since baseline visit; unk = unknown
DREXEL MEDICINE
10. Association analysis of HIV-1 LTR SNPs with regard to change in
CD4 count and change
Phenotype Position Location Ref/Mut Mutant Frequncy Effect p-value
๏CD4 273 unk C/A 8.3% -42.705 0.0486
304 unk T/A 13.2% -38.809 0.0336 Effect = change in
314 unk G/ACT 6.8% -60.034 0.0162
381 Sp III (5) C/T 27.9% 31.887 0.0301
CD4 count between
385 Sp III (9) G/A 8.3% -57.781 0.0215 visits
398 unk T/CGA 21.4% -42.769 0.0122
๏Log (Viral) 108 COUP/Ap 1 A/CGT 37.3% 0.161 0.0157
USF
Effect = % change
290 C/T 6.4% 0.319 0.0191
383 Sp III (7) T/C 6.0% 0.302 0.0425 in VL between visits
adjusted for sex, age, race, days since baseline visit; unk=unknown
DREXEL MEDICINE
11. Electrophoretic mobility shift assay (EMSA)
3. Run native PAGE
2. Incubate DNA probe with protein (N.E.) DNA
(probe)
antibody
TF
32P
TGACTCA
32P
1. Label double-stranded DNA
(oligonucleotide with binding element)
32P
TGACTCA
32P
12. Disease severity of patient 107 increases over the four visits
Li and Aiamkitsumrit et al 2011
DREXEL MEDICINE
13. Patient acquires neurocognitive impairment over time
Patient Neurologic Neurologic Neurologic
Visit score test complications
A0107- depression, bipolar,
normal MSK
R00 schizophrenia
A0107- modified depression, bipolar,
12
R01 Hopkins schizophrenia
A0107- modified depression, bipolar,
8
R02 Hopkins schizophrenia
A0107- modified depression, bipolar,
9.5
R03 Hopkins schizophrenia
A0107-R01 A0107-R02 A0107-R03
Score: 2/2 Score: 0/2 Score: 0/2
Li and Aiamkitsumrit et al 2011
DREXEL MEDICINE
14. Status of amplification and cloning of patient 107 tat genes
Isolation of
Patient Growth of Round 1 Round 2 pcDNA Sequence
4.4KB
Visit Culture Amplification Amplification Hygro3.1 Confirmed
Fragment
R00 โ โ โ โ ?
R01 โ โ โ โ ?
R02 โ โ ?
R03 โ โ ?
R04 โ โ ?
R05 โ โ ?
R06 โ โ ?
DREXEL MEDICINE
16. Majority of 4.4 kb insert lost during sample preparation
GAG-POL ENV
tm
LTR pr rt in VPR su (gp 120)
(gp 41)
GAG VIF tat NEF LTR
rev
T-7 Primer M13R Primer
4.4kb
DREXEL MEDICINE
17. Domains of Tat and important amino acid motifs
Basic
Cysteine-rich domain/TAR
Acidic domain domain Core binding EXON 1 EXON 2
domain/ARM
21 22 30 37 38 48 49 57 58 72 73 101
C C C CC C C LGISYG RKKRRQRRR RGD KKK E E E
I II III IV V VI
Basic region 86 87
Nuclear
Transactivation/ localization
CycT1 binding C/EBP
Co-factor binding binding
CBP/p300
Sp1
binding DNA-PK
binding
18. DREXELMED Cohort Tat sequences show conserved
cysteine residues and position 100 variation
Tat AA Position 100 analysis
Pt ID R00 R01 R02 R03 R04 R05 R06
Time (mnths) between
9 10 8 17 6
visits
37
Neuro NA NA 3.5 ND 10 11
Tat V to H
Time (mnths) between
9 10 9 5 18 5
visits
41
Neuro NA NA 4.5 8 5.5 ND ND
Tat V to F
Time (mnths) between
23
visits
51
Neuro norm 9
Tat conB
Time (mnths) between
19 8 12 18
visits
56
Neuro sub 6 9 9
Tat V to C
Time (mnths) between
14 2 11 6 3 5
visits
107
Neuro norm 12 8 9.5 10 11.5 10.5
Tat V to K V to K
Time (mnths) between
36
visits
119
Neuro norm 5
Tat conB
Time (mnths) between
visits
131
Neuro 4
Tat V to I
Time (mnths) between
visits
220
Neuro 11
Tat 3 conB
19. Brian Wigdahl, Ph.D., Professor & Chair
Department of Microbiology & Immunology
Drexel University College of Medicine
Edward Archaempong, Ph.D. Sonia Navas-Martin, Ph.D. Adriano Ferrucci Liudmila Mazaleuskaya
Jeffrey Jacobson, M.D. Michael Nonnemacher, Ph.D. Archana Gupta Saifur Rahman
Pooja Jain, Ph.D. Vanessa Pirrone, Ph.D. Bryan Irish Viraj Sanghvi
Steve Jennings, Ph.D. Laura Steel, Ph.D. Shawn Keogan Sonia Shah
Zafar Khan, Ph.D. Nirzari Parikh, M.S. Christina Kollias Luz Jeanette Sierra
Sandhya Kortagere, Ph.D. Shendra Passic, M.S. Jason Lamontage Melany Simmons
Fred Krebs, Ph.D. Benjamas Aiamkitsumrit Luna Li Marianne Strazza
Michele Kutzler, Ph.D. Brandon Blakely Karissa Lozenski Ken Thompson
Julio Martin-Garcia, Ph.D. Sharon Bandstra Raphael Lukov Kamiliah Williams
Brian Moldover, Ph.D. Betty Condran Sharron Manuel Adam Wojno
Olimpia Meucci, M.D., Ph.D. Satinder Dahiya Nyree Martin
NINDS NIMH NCI NIDA NIAID CONRAD
DREXEL MEDICINE
Editor's Notes
Proviral DNA is integrated in host chromosome is approximately 9.6 kb in length, which encodes for a number of HIV proteins. Important for todayโs talk is that it encodes for the HIV regulatory protein Tat. The transcription, which is driven by RNA pol, occurs through the interaction of this enzyme with the viral promoter, or long terminal repeat. Contained in the 4.4 kb segment is the vpr region, tat, envelope, nef, and the LTR.
Basal HIV transcription occurs through the interaction of RNA pol II enzyme with various cellular transcription, principal being NF kappa-B factors. This basal transcription results in short and long transcipts of the HIV genome. Important for the talk today is the fact that these transcripts encode for Tat and it is the build up of this protein that leads to superactivation of HIV transcription.
Thatsuperactivation is depicted here, where you can see Tat interacting with the TAR element, allowing for increased processivity and elongation of the RNA pol II. This leads to between a 30 and 70X increase in transcription.
So, my PI and I have developed a hypothesis that states that genetic variation within HIV-1 LTR and Tat co-select due to selective pressures applied to HIV genome during replication, resulting in altered function of the LTR, Tat, or both. If anyone asks: reverse transcriptase is driving the SNP changes that we see in the genome.
SNPs associate with CD4 T cell count and viral load. Scatter plot of p-valuesUsing the preliminary data from 350 subjects with a total of 602 visits, the results of the best fit models demonstrated both gender (5x10-5)and time (days since first visit, p=0.0021) are significantly associated with CD4 counts. Age, cocaine use, and time are all significant (0.0024, 0.0041, 1x10-7, respectively) in affecting viral load while gender is not. Cocaine users have 2.74 times more viral load than non-users. In a preliminary SNP association study, SNPs identified above were associated with CD4 T cell count and viral load. We have found 7 SNPs significantly associated with CD4 and 3 with viral load at p=0.05, after adjusting for age, gender, cocaine use, and time (Fig. 6).
As an initial investigation of CD4 and viral load progression, the association between the SNPs and relative changes of CD4 and viral load at following visits compared to their baseline levels using the same models were tested (Fig. 6). 8 SNPs were found significant and 6 of them are associated with greater changes of CD4 and viral load. The possible associations between these SNPs and the relative change confirm our hypothesis that SNPs may affect disease progression and prompt examination of the next hypothesis regarding progression rate and SNPs.
What was interesting about this patient is that the 3T/5T variation existed as a predominant quasispecies within the patient when she had a good CD4 count and low viral load.
Even more interesting was that these variants existed prior to this patient declining in their ability to perform well on a mini-neurologic exam. This mini-neurologic exam tests a patient for psychomotor speed, memory recall, and constructional abilities. A perfect score on this test is a 12; any score below 10 is considered neurologically impaired. As you can see on the right table, as this patient came back for subsequent visits, her neurologic score declined. In addition to the neurologic score decline, the important point to notice in that in the previous slide, her CD4 count declined. While this patient can be viewed as a proof of concept for the original 3T/5T observation, the lab was interested in understanding the functionality of the LTR and Tat from this patient and if it potentially contributed to the increase in pathogenesis.
Current progress to date includes growing up cultures of bacteria into which the HIV genome has already been cloned from patient 107 blood samples. I have been able to grow up clones from all patient 107 visits, and isolate the 4.4KB fragment from the genome. However, this process has not been without its problems.
One such portion of the genome which is amplified is called the 4.4 kb fragment. This fragment spans from the VPR to the 3 prime LTR. As you can see, within this fragment, the Tat gene is contained. Important to note is that Tat is a protein that is made through two exons which are spliced together (keep this in mind, as it will be important in describing some of the techniques that I am employing to assist this project).
Tat has been divided into six different functional domains. The two domains that are of importance to this presentation are 2 and 6. While I am bringing these two domains to your attention, I performed sequence analysis on all domains to look for amino acid changes that were common to patients that demonstrated neurocognitive impairment. In regards to domain 2, we found that the cysteine rich region was conserved in all patients through all samples obtained. Domain 6, however, did show some changes that correlated with neurocognitive impairment. The two important changes that were seen in patients that did demonstrate neurocognitive impairment were residue changes in both positions 74 and 100, both located in exon II domain 6 of the protein, the domain that plays a role in viral infectivity, cell surface integrin binding and NF-kappa-B transactivation of replication. Interestingly enough, both of these changes correlated with brain derived Tat.
When you look at position 100 specifically, you will notice that during the second visit, labeled R01, this change was detected. During subsequent visits in patients 37, 41, and 107, neurocognitive assessments scores were suboptimal, indicating neurocognitive impairment. For patient 56, the change was detected during the first visit, with neurocognitive impairment detected in all subsequent visits.