What are the responsibilities and job description for the Biologist 2 position at ORAU?
Overview:
Project Background:
A preferred Ph.D. degree in any branch of Life Science with the following essential preferred criteria:
ORAU is seeking a Biologist to assist the Federal Drug Administration (FDA) with a project for up to 5 years. The successful candidate will become a temporary employee of ORAU and will be placed at FDA in Silver Spring, MD. Total compensation includes a competitive salary, healthcare benefits, Paid Time Off, and paid holidays!
Project Background:
Prior research in FDA’s laboratory found that genes involved in cellular metabolism, apoptosis, immune cell proliferation and activation, cytokines, chemokines, and transcription factors were differentially expressed in HIV-1 infected PBMCs. Contrary, relatively few genes were differentially expressed in PBMCs infected with HIV-2 (Devadas et al., PLoS ONE 2016 11(1): e0147421. doi: 10.1371/journal.pone.0147421). Dr. Hewlett's laboratory also used microarrays to analyze expression profiles of lincRNAs and mRNAs in monocyte-derived macrophages (MDMs) infected with HIV-1/HIV-2. Our study identified many differentially expressed lincRNAs and mRNAs in MDMs infected with HIV-1/HIV-2 compared to uninfected MDMs. Genes involved in glutathione metabolism and lysine degradation were differentially regulated only in HIV-1 infected MDMs. In HIV-2 infected MDMs, CUL 2, SFRS9, and RBBP4 genes were differentially expressed. Furthermore, we found that plasma levels of lincRNA: chr2: 165509129-165519404 and lincRNA: chr12: 57761837-57762303 were better indicators of HIV-1 infection while lincRNA: chr10:128586385-128592960, XLOC_001148, and lincRNA: chr5:87580664-87583451 were better indicators of HIV-2 infection. (Scientific Reports | (2018) 8:2546 | DOI:10.1038/s41598-018-20791-6).
In addition, the differential expression profiles of host micro RNAs (miRNA) in cells infected with HIV-1 and HIV-2 were analyzed using miRNA profiling PCR arrays.
Differentially expressed miRNAs were identified, and their putative functional targets were identified. The results indicate that hsa-miR 541-3p, hsa-miR 518f-3p, and hsa-miR 195-3p were consistently up-regulated only in HIV-1 infected cells. The expression of hsa-miR 1225-5p, hsa-miR 18a*, and hsa-miR 335 were down modulated in HIV-1 and HIV-2 infected cells. Putative functional targets of these miRNAs include genes involved in signal transduction, metabolism, development, and cell death. (Viruses 2016, 8, 121; doi:10.3390/v8050121)
The research indicates that coding and non-coding RNA were differentially expressed in HIV-1 and HIV-2 infected PBMCs. The project's goal is to develop a signature of biomarkers based on mRNA and non-coding RNA by using comparative genomics-based profiling of T cells. Monocyte-derived macrophages (MDMs) infected with HIV-1, HIV-2, and other HIV-1 subtypes, and to evaluate the potential of these novel biomarkers to distinguish acute vs. chronic phase of infection. Utilizing these biomarkers, we aim to study the pathogenesis specific to the subtype virus and identify potential new targets for therapy. This work involves: testing plasma from HIV-infected individuals, isolating the virus, host mRNA, host miRNA, and non-coding RNA from plasma, followed by absolute quantification by digital droplet PCR; microarray/PCR-array to identify differential RNA expression, validate host genes to establish a signature of biomarkers, and study pathogenesis by overexpression/knockdown of these biomarker targets; and use of ddPCR for quantifying gene expression and viral load from different samples.
Responsibilities:
Tasks:
- Quantitate viral load in FDA panels using digital droplet PCR (ddPCR)
- Isolate, propagate and maintain primary cells PBMCs and monocyte-derived macrophages
- Propagate and maintain A549, Jurkat, CEM, U937, HeLa, Ghost, ACH2, and U1 cell lines
- Infect primary cells and cell lines with HIV-1, HIV-2, and HSV2 viruses
- Extract, purify, and manipulate nucleic acids.
- Transfect cells with lentiviral and adenoviral vector constructs for expressing host genes
- Transfect cells with shRNA constructs
- Analyze transfected cells for gene expression and knockdown of genes
- Run qPCR, droplet digital PCR (ddPCR), and PCR arrays for identifying expression of host genes, miRNA, tRNA, and non-coding RNA
- Identify a panel of biomarkers to distinguish acute vs. chronic phases of HIV-1 infection.
- Design and synthesis of primers to amplify and quantitate HIV-1 and HIV-2 RNA in digital droplet PCR
- Identify differences in the expression of circulating host non-coding RNA biomarkers in females vs. males to detect early HIV-1 infection.
- Identify differences in the expression of host non-coding RNA biomarkers in response to PrEP and other anti-retroviral treatment Interventions in males vs. females using genomics, proteomics, and NGS.
- Validate the identified biomarkers using TaqMan and immuno-techniques in clinical samples to develop a panel of non-coding RNA that can be used as biomarkers to detect early HIV-1 infection when viral markers are not detectable
- Evaluate the identified biomarkers using techniques like real-time PCR, ddPCR, next-generation sequencing, EMSA, Western blotting, ELISA, Luminex assays, FACS, and fluorescence microscopy
- Analyze data using Instat GraphPad, ImageJ, Ingenuity Pathway Analysis, Cytoscape, SPSS
Deliverables:
- A bi-weekly report presentation to the principal investigator of the ongoing experiments
- Design and synthesize primers for amplification and quantification of HIV-1 and HIV-2 RNA in digital droplet PCR (5th month)
- Provide raw data (real-time PCR report) evaluating biomarkers (host genes, miRNA, tRNA, and non-coding RNA) involved in HIV pathogenesis and infection (12th month)
- Provide data (non-coding RNA expression data) from HIV-1 infected males and females (12th month)
- Provide supporting data (real-time PCR report) identifying signature biomarkers to distinguish acute vs. chronic phase of HIV-1 infection (5th month)
- Analyze data using Instat GraphPad, ImageJ, Ingenuity Pathway Analysis, Cytoscape, SPSS and provide data for use in a publication in peer-reviewed journals by the end of this appointment
- Present research findings in group meetings (quarterly), division research seminars (annually), and other scientific conferences (annually)
A preferred Ph.D. degree in any branch of Life Science with the following essential preferred criteria:
- Eleven or more years of training in the safe handling of human blood samples
- Six or more years of research experience in HIV-1 and HIV-2 research
- Good practice of work in Biosafety Level 2 plus and Level 3 laboratories
- Experience with analysis of gene expression data from microarray platforms for identification of HIV related biomarkers
- Proficiency in basic virological techniques such as virus propagation, viral load testing using digital droplet PCR, and HIV-1/HIV-2 isolation from cell culture and different body fluid compartments
- Experience with cell culture techniques such as primary cell isolation, cell line propagation and maintenance, infection with different types of viruses, transfection of primary cells and cell lines
- Experiencing with grant writing
- Broad knowledge of techniques that may be applied to study host factor and virus pathogenesis, such as tissue culture, flow cytometry, extraction and purification of nucleic acid, quantitative RT-PCR, lincRNA quantification, and protein-based methods such as Western blotting and Luminex assays
- Experience using Microsoft Office 2010, Instat GraphPad, ImageJ, Ingenuity Pathway Analysis, Cytoscape, SPSS, and EndNote software.
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