genomic and proteomic approaches

what is the goal of using genomic and proteomic approaches to study host-pathogen interactions

to investigate how pathogens interact with host cells on a molecular level, using large-scale genomic and proteomic data to understand these interactions

what are the main molecular levels studied in host-pathogen interactions

protein-gene interactions (genome), protein-protein interactions (proteome), and biochemical reactions (metabolome)

what is the citrate cycle, and how is it related to the metabolome

a series of biochemical reactions that produce energy in cells, part of the broader study of the metabolome

what is co-immunoprecipitation (COIP) used for in proteomics

used to detect protein-protein interactions by lysing cells, denaturing proteins, and separating them based on size

what are some methods for detecting protein interactions

include Yeast Two-Hybrid (Y2H), NAPPA, LUMIER, PCA, MAPPIT, and TAP

what is the Yeast Two-Hybrid (Y2H) system used for

used to detect protein-protein interactions by introducing recombinant genes encoding target (bait) and binding (prey) proteins into yeast cells.

how does the Y2H system work

ccombines a DNA binding domain (bait) and a transcriptional activation domain (prey), if bait and prey proteins interact, they activate the transcription of a reporter gene, signaling interaction

what was the Y2H system used to investigate in the study mentioned

system was used to screen for influenza virus-host protein interactions across six strains of influenza and five libraries, leading to the discovery of 472 virus-host protein interactions

how many hits did the Y2H matrix screen for influenza virus-host protein interactions produce

screen produced 2,498 hits, which were narrowed down to 154 high-confidence interactions

what is one weakness of large-scale interaction screens like Y2H

they can produce false positives, so further validation is needed to confirm true interactions.

why is validation important in protein interaction studies

eliminate false positives and ensure that the identified interactions are biologically relevant

what is the proteome

entire set of proteins expressed by a genome, tissue, or organism at a certain time

what is the genome

complete set of genetic material (DNA) in an organism

what is the purpose of analyzing large-scale genomic data

understand the complex interactions between host and pathogen at a molecular level, which can reveal new therapeutic targets

what are the strengths of proteomic approaches like Y2H

can identify protein-protein interactions on a large scale, revealing complex interaction networks between host and pathogen

what is a pathogenicity island in terms of plasmid/chromosome organization

cluster of genes on a plasmid or chromosome encoding proteins that contribute to a pathogen's ability to cause disease

what are the advantages of Yeast Two-Hybrid (Y2H) screens for detecting protein interactions

quick, inexpensive, and fast

what are the disadvantages of Y2H screens

they can produce false positives (low specificity) and false negatives (low sensitivity)

how can the coverage of Y2H screens be improved

addressing the "major drift" between large tags and small peptide tags, ensuring both are correctly folded and functional

what role do chaperones play in protein folding during Y2H assays

help proteins fold correctly, especially domains that fold independently

how are protein interactions validated using LUMIER assays

involve tagging proteins with luciferase to measure their binding interactions in live cells

what is the principle behind the protein complementation assay (PCA")

detects protein interactions based on the reconstitution of a fluorescent signal (e.g., YFP) when two protein fragments (C-terminal and N-terminal) are brought together

what is the excitation and emission wavelength of YFP in PCA

excitation occurs at 500 nm, and emission is detected at 530 nm

what happens if the C-terminal and N-terminal fragments of YFP are expressed independently in PCA

do not emit fluorescence unless they interact, confirming protein-protein interactions

in PCA of VZV ORF24N and ORF27, what was discovered in HeLa cells

ORF24N interacts with ORF27 in the nucleus, suggesting a role in viral nuclear functions.

what is an ORF (Open Reading Frame)

continuous stretch of codons that encode a protein, starting with a start codon and ending with a stop codon.

what is an advantage of PCA in visualizing protein interactions

can localize interactions within specific cellular compartments, such as the nucleus

what type of microscopy is used to observe cellular interactions in PCA assays

used to track protein interactions and identify where in the cell these interactions occur

what is the first step in mass spectrometry for protein identification

small sample is ionized, typically forming cations by the loss of an electron.

what occurs in the Mass Analyzer of mass spectrometry

ions are sorted and separated based on their mass-to-charge ratio (m/z)

what happens after ions are sorted in mass spectrometry

the separated ions are measured, and the results are displayed on a chart or spectrum

what is a functional (perturbation) screen in the context of protein identification

method to identify the function of proteins by disrupting their activity using techniques like RNA interference

what is a genome-wide RNA interference screen

high-throughput technique that uses pooled siRNA to systematically knock down genes across the genome to study their effects

how are HeLa cells involved in RNA interference screens

HeLa cells are often used for high-throughput reverse transfection with siRNA to assess the impact on cell functions, such as virus replication

what does the HSV-GFP infection assay measure

measures the growth curve of virus replication by detecting GFP fluorescence as an indicator of infection

what is the purpose of a Cell Viability assay in RNA interference screening

assess whether the siRNA treatments affect the survival of the cells, indicating the effectiveness of gene knockdown

what occurs in the Secondary Screen of RNA interference screening

validation of the primary screen results using 4 siRNAs per gene to confirm hits and perform functional validation assays

what is CRISPR/Cas9 gene editing used for in protein identification

employed to create precise gene knockouts, allowing researchers to study the effects of specific gene deletions on protein function

what are haploid genetic screens

utilizes haploid organisms to facilitate the identification of gene functions by eliminating redundancy in genetic interactions

what is Next Generation Sequencing (NGS)

modern sequencing technology that allows rapid sequencing of entire genomes, enabling comprehensive analysis of genetic materia

what is the purpose of Whole Genome Sequencing

determine the complete DNA sequence of an organism’s genome, providing insights into genetic variations and potential gene functions

what is cDNA microarray technology

high-throughput method used to measure the expression levels of many genes simultaneously by hybridizing complementary DNA (cDNA) to arrayed probes on a glass slide.

what is the role of reverse transcription in transcriptomics

reverse transcription is the process of converting RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase, which is essential for cDNA microarray analysis.

what is hybridization in the context of cDNA microarrays

the process where cDNA binds to complementary DNA probes on the microarray, allowing the detection of specific gene expressions based on the binding of fluorescently labeled cDNA.

what do the colors represent in microarray results

• Red indicates upregulation of genes (higher expression in one sample).

• Green indicates downregulation (lower expression).

• Black indicates constitutive expression (no change in expression levels)

what is the significance of laser emission in analyzing cDNA microarrays

laser scans the microarray to excite the fluorescent dyes, enabling detection and quantification of the bound cDNA based on emitted light, which correlates with gene expression levels

what is a dye-specific hybridization (HZ) in microarray analysis

refers to using different fluorescent dyes for distinct samples, facilitating comparative analysis of gene expression across multiple conditions

what is systems discovery in the context of macrophages

involves using high-throughput technologies and computational analysis to understand the complex interactions and pathways activated in macrophages during infection or treatment.

what role do macrophages play in the immune response to MCMV infection

key immune cells that recognize and eliminate MCMV-infected cells through phagocytosis and by producing inflammatory cytokines.

hw does IFN-gamma (IFNG) affect macrophages

potent cytokine that activates macrophages, enhancing their antimicrobial activities, promoting antigen presentation, and increasing the production of reactive nitrogen and oxygen species

what are the differences between immune-activated macrophages and those that are viral-infected

show enhanced expression of genes related to inflammation and pathogen clearance, while viral-infected macrophages may exhibit altered gene expression patterns that reflect viral replication and immune evasion strategies

what are some methods used to study the effects of IFNG and MCMV on macrophages

include gene expression profiling (e.g., RNA-seq), protein analysis (e.g., mass spectrometry), flow cytometry for cell surface markers, and cytokine assays to measure immune responses

how can pathway analysis contribute to understanding the effects of IFNG and MCMV in macrophages

helps identify key signaling pathways and molecular interactions activated in macrophages, allowing for a deeper understanding of the immune response during infection and treatment