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I was going through this paper, but did not understand a term.
What is the meaning of functional screen?
(I am not a biology student, I don't understand much, and a small simple explanation would be enough)
From the abstract of the linked article (Guttman, 2002):
We used an in vivo genetic screen to identify 13 effectors [… ]. Although sharing little overall homology, the amino-terminal regions of these effectors had strikingly similar amino acid compositions.
And from the body:
The screen relied on the type III secretion signal and the endogenous promoter of the hop gene and was thus highly specific.
Hence, the authors search and identify proteins with a secretory signal under the regulation of a hop promoter. A functional screen in this paper refers to the analysis of protein samples to detect the presence of proteins with a particular function.
Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens
Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell.
Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes.
Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.
A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence
Primary fibroblasts respond to activated H-RAS V12 by undergoing premature arrest, which resembles replicative senescence 1 . This irreversible 'fail-safe mechanism' requires p19 ARF , p53 and the Retinoblastoma (Rb) family: upon their disruption, RAS V12 -expressing cells fail to undergo senescence and continue to proliferate 1,2,3,4,5,6,7 . Similarly, co-expression of oncogenes such as c-MYC or E1A rescues RAS V12 -induced senescence. To identify novel genes that allow escape from RAS V12 -induced senescence, we designed an unbiased, retroviral complementary DNA library screen. We report on the identification of DRIL1, the human orthologue of the mouse Bright and Drosophila dead ringer transcriptional regulators. DRIL1 renders primary murine fibroblasts unresponsive to RAS V12 -induced anti-proliferative signalling by p19 ARF /p53/p21 CIP1 , as well as by p16 INK4a . In this way, DRIL1 not only rescues RAS V12 -induced senescence but also causes these fibroblasts to become highly oncogenic. Furthermore, DRIL1 immortalizes mouse fibroblasts, in the presence of high levels of p16 INK4a . Immortalization by DRIL1, whose product binds the pRB-controlled transcription factor E2F1 (ref. 8), is correlated with induction of E2F1 activity. Correspondingly, DRIL1 induces the E2F1 target Cyclin E1, overexpression of which is sufficient to trigger escape from senescence. Thus, DRIL1 disrupts cellular protection against RAS V12 -induced proliferation downstream of the p19 ARF /p53 pathway.
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The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein–DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein–DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency.
Chromatin Immunoprecipitation-Based Screen To Identify Functional Genomic Binding Sites for Sequence-Specific TransactivatorsFIG. 1 . Analysis of p53, MDM2, and p21 protein levels in MCF-10A cells and HMEC after ADR treatment. Shown is Western blot analysis of p53, MDM2, and p21 protein harvested from MCF-10A and primary HMEC that were not treated (−) or treated (+) with ADR (350 nM) for 8 h. FIG. 2 . Yeast selection system. The candidate upstream activating sequences (UAS) recovered from ChIP were cloned into the pBM947 reporter vector containing the HIS3 gene under the control of a basal GAL1 promoter and a URA1 marker. The pBM947-based library was transformed into an auxotrophic His-deficient yeast strain containing the pRS314SN vector, which expresses a galactose-inducible human wild-type p53 and a TRP1 marker. Yeasts containing both the vectors were grown on galactose-containing, histidine-deficient media (SG-Trp-Ura-His) to assay for the ability of p53 to bind to the potential UAS in the pBM947 vector and activate transcription of the HIS3 gene. Replica plating of all clones on glucose-containing, histidine-deficient media (SD-Trp-Ura-His) was performed to rule out false-positive clones. The clones that grew in the presence of glucose were considered false positive, and only the clones that grew on galactose, and presumably in a p53-dependent manner, were analyzed further. A clone containing a fragment of the p21 promoter encompassing site 1 is indicated as an example of a positive result. FIG. 3 . Analysis of p53 in vivo binding to consensus binding sites. Three sets each of MCF-10A cells, HMEC, and HK cells were identically processed: one set was treated with ADR (350 nM for 5 h) and formaldehyde cross-linked (ADR +, X-L +), another set was not treated with ADR and was formaldehyde cross-linked (ADR −, X-L +), and a final set was treated with ADR and not formaldehyde cross-linked (ADR +, X-L −). The DNA for PCRs was derived from p53-specific and cyclin B1-specific immunoprecipitations (IP) and amplified using primers flanking the p53 response elements in genes encoding the indicated proteins. PCRs were resolved with polyacrylamide gel electrophoresis, and the gels were stained with ethidium bromide. The cyclin B1-specific IPs were included to assess any DNA fragments purified from cross-linked lysates nonspecifically. Input +, genomic input input −, water control. PCR results with primers directed to the coding region of GAPDH serve as a control for nonspecific DNA IP by p53-specific antibodies. FIG. 4 . Comparative analysis of p53 binding to sites in promoter regions of known and candidate target genes. In the left panels, four sets of HMEC were processed as follows: one set was treated with ADR (350 nM for 5 h), formaldehyde cross-linked, and immunoprecipitated with a p53 antibody (solid bars) another set was treated with ADR, formaldehyde cross-linked, and immunoprecipitated with a cyclin B1 antibody (open bars) a third set was treated with ADR, not formaldehyde cross-linked, and immunoprecipitated with a p53 antibody (dotted bars) and a final set was formaldehyde cross-linked but not treated with ADR and then immunoprecipitated with a p53 antibody (gray bars). Quantitative real-time PCR was performed, and each sample was normalized to the same genomic DNA that was isolated from cells that were cross-linked and processed the same with the exception that the immunoprecipitation step was not performed. The binding sites shown are those that were recovered from the library screen (LS), those that were previously reported in the literature (RS), and those that were potential binding sites found by gene analysis using the p53MH algorithm (PS). The base pair match of the binding site to the p53 consensus is shown in parentheses. The results, shown as percentages of input DNA, are from at least three independent experiments, with the error bars representing standard deviations. The right panels show schematics of the genomic structure and localization of known and putative p53 binding sites analyzed. The bar shading indicates species conservation as indicated. Exons are indicated with an E followed by the exon number in either an open box or a shaded box (representing the terminal exon). The sequences of the binding sites present in the regions analyzed are shown, and in parentheses the distances of the binding sites from the start of exon 1 are given. FIG. 5 . p53-dependent regulation of representative candidate target gene expression. The isogenic pair of HCT116 p53 +/+ and p53 −/− cells were treated with ADR (350 nM) for 0, 6, 12, and 24 h the HIp53 cells (p53) and corresponding vector control cell line (Ø) were treated with ponasterone A (10 μM) for 24 h, and the HK cells were infected with a GFP- or p53-expressing adenovirus for 30 h. Total RNA from HCT116 cells and mRNA from HIp53 and HK cells was purified and reverse transcribed and quantitative real-time PCR performed. The samples were normalized to GAPDH, and the results are presented as changes relative to either the 0-h HCT116 p53 +/+ sample (left panel), the HIp53 vector control cells treated with ponasterone (middle panel), or the HK cells infected with a GFP-expressing adenovirus (right panel). The results are the means of three independent experiments (MCF-10A cells and HMEC) or duplicate experiments (HK cells), with error bars representing the standard deviations. Note that the y axes are set to 7.0 with the exceptions of the left and middle panels for the CDKN1A gene and the left and right panels for the EDN-2 gene.
A functional specification does not define the inner workings of the proposed system it does not include the specification of how the system function will be implemented. Instead, it focuses on what various outside agents (people using the program, computer peripherals, or other computers, for example) might "observe" when interacting with the system.
A functional requirement in a functional specification might state as follows:
When the user clicks the OK button, the dialog is closed and the focus is returned to the main window in the state it was in before this dialog was displayed.
Such a requirement describes an interaction between an external agent (the user) and the software system. When the user provides input to the system by clicking the OK button, the program responds (or should respond) by closing the dialog window containing the OK button.
There are many purposes for functional specifications. One of the primary purposes on team projects is to achieve some form of team consensus on what the program is to achieve before making the more time-consuming effort of writing source code and test cases, followed by a period of debugging. Typically, such consensus is reached after one or more reviews by the stakeholders on the project at hand after having negotiated a cost-effective way to achieve the requirements the software needs to fulfill.
- To let the developers know what to build.
- To let the testers know what tests to run.
- To let stakeholders know what they are getting.
In the ordered industrial software engineering life-cycle (waterfall model), functional specification describes what has to be implemented. The next, Systems architecture document describes how the functions will be realized using a chosen software environment. In non industrial, prototypical systems development, functional specifications are typically written after or as part of requirements analysis.
When the team agrees that functional specification consensus is reached, the functional spec is typically declared "complete" or "signed off". After this, typically the software development and testing team write source code and test cases using the functional specification as the reference. While testing is performed, the behavior of the program is compared against the expected behavior as defined in the functional specification.
One popular method of writing a functional specification document involves drawing or rendering either simple wire frames or accurate, graphically designed UI screenshots. After this has been completed, and the screen examples are approved by all stakeholders, graphical elements can be numbered and written instructions can be added for each number on the screen example. For example, a login screen can have the username field labeled '1' and password field labeled '2,' and then each number can be declared in writing, for use by software engineers and later for beta testing purposes to ensure that functionality is as intended. The benefit of this method is that countless additional details can be attached to the screen examples.
We thank Hans Teunissen and Elzo de Wit for their help on the 3C experiment and all members of the Agami laboratory for their technical help and discussions. We are grateful to the NKI Genomics Core Facility for deep-sequencing our samples.
This work was supported by the ERC-AdG enhReg (322493 to RA), ERC-ITN RNA TRAIN (607720 to RA), China Scholarship Council (CSC) (to LL), The Human Frontier Science Program LT000640/2013 (to APU), and The Dutch Organization for Research NWO-TOP 91216002 (to RA). RE is supported by the Israeli Cancer Association (ICA), with the generous assistance of the ICA Netherlands friends, and by the Marguerite Stolz Research Fellowship Fund. ZM was supported in part by the Gad, Nava, and Shye Shtacher fellowship. RE is a Faculty Fellow of the Edmond J. Safra Center for Bioinformatics at Tel Aviv University.
Availability of data and materials
RNA-seq data are available from the GEO DB accession number GSE112458 . GRO-seq data are available from the GEO DB accession number GSE109290 .
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Anatomy of a molecule: What makes remdesivir unique?
The World Health Organization in late January convened experts to discuss experimental therapeutics for patients with the emerging coronavirus with no name, no vaccine and no treatment. The panel reported that &ldquoamong the different therapeutic options, remdesivir was considered the most promising candidate.&rdquo
Within weeks, a clinical trial of the compound was underway in China. Results are expected in April in the meantime, the outbreak of SARS-nCoV-2, the virus that causes COVID-19, has become a global pandemic.
Remdesivir is a nucleoside analog, one of the oldest classes of antiviral drugs. It works by blocking the RNA polymerase that coronaviruses and related RNA viruses need to replicate their genomes and proliferate in the host body.
The molecule originally was synthesized as part of a screen for inhibitors of the hepatitis C virus RNA polymerase. Its inventors at Gilead Sciences decided to move forward with a different nucleoside analog compound to treat hepatitis C. But RNA-dependent RNA polymerases are conserved between many viruses. Experiments in vitro, in cell culture and in animal models have shown that remdesivir has broad-spectrum activity against RNA viruses, including filoviruses (like the one that causes Ebola) and coronaviruses.
Remdesivir resembles the RNA base adenosine, shown here as a monophosphate.
The compound and ATP have some important differences, but some features are very similar. ASBMB Today spoke to medicinal chemist Katherine Seley&ndashRadtke at the University of Maryland, Baltimore County, and structural virologist Craig Cameron at the University of North Carolina, Chapel Hill about what makes the molecule interesting. Click on a feature marked in blue to read their remarks.
3&rsquo hydroxy group
Different classes of nucleoside/nucleotide analogs have different effects on polymerases. Remdesivir is in a class called nonobligate chain terminators, because it should, in theory, be possible to add more nucleotides to a strand of RNA after remdesivir has been added due to the presence of the hydroxyl group at carbon 3 in the sugar.
&ldquoThat hydroxy group is what is required for continued synthesis of nucleic acid, whether it be RNA or DNA,&rdquo said virologist Craig Cameron, a professor at the University of North Carolina at Chapel Hill who studies the interactions between nucleoside analogs and viral polymerases.
Recent research suggests that when mixed with RNA polymerases from coronaviruses or flaviviruses in vitro, remdesivir doesn&rsquot terminate the synthesis of a new RNA strand right away. Instead, Cameron said, &ldquoIt takes a few cycles of nucleotide addition before you can see the termination effect.&rdquo
Those additional nucleotides may help shield remdesivir from coronavirus proofreading enzymes that are known to remove unnatural nucleotide analogs.
Base pairing to uracil
In adenosine in double-stranded RNA, this face of the molecule is involved in base-pairing with uracil. The two nitrogens act as proton donor and acceptor, respectively for hydrogen bonds to atoms in the uracil base.
Chemists think that remdesivir, by presenting a very similar binding face, gets incorporated into a growing RNA strand by viral polymerases.
The link between ribose and the base is called the glycosidic bond. Usually, it connects the 1&rsquo carbon in the ribose ring to a nitrogen in the base. But in remdesivir (and some other nucleotide analogs) the sugar and the nucleobase are connected by a bond between two carbons.
&ldquoIt definitely provides much greater stability (against) nucleases and other enzymes that can cleave the nucleobase from the sugar,&rdquo said Katherine Seley-Radtke, a medicinal chemist at the University of Maryland, Baltimore County who works on the design and synthesis of antiviral nucleoside analogues. With a C-nucleoside, &ldquoyou&rsquod have to break a carbon-carbon bond, whereas in a normal nucleoside you&rsquore breaking a hemi-aminal bond, which is actually fairly unstable. So having that carbon-carbon bond is a great advantage.&rdquo
1&rsquo cyano group
Ask a group of chemists what jumps out at them about remdesivir, and most will start with this dramatic feature. Substitution at this carbon is unusual, and probably only possible because of the strength of the C-nucleoside bond.
According to an article in the Journal of Medicinal Chemistry, the cyano group was initially added because a precursor molecule, a very effective inhibitor of viral RNA polymerases, also blocked the mitochondrial RNA polymerase in mice. To make a molecule without those toxic side effects, chemists at Gilead tried a series of substitutions at the 1&rsquo carbon. The compound with the cyano group worked best: it still blocked the hepatitis C polymerase, but was no longer incorporated by host cell polymerases.
&ldquoYou can&rsquot predict activity. You have to make it and test it,&rdquo Seley-Radtke said. &ldquoBut even small changes can have amazing consequences.&rdquo
&ldquoYou see all that flotsam and jetsam coming off at the 5&rsquo hydroxyl?&rdquo said Katherine Seley-Radtke. Among medicinal chemists, this type of protecting group is casually known as &ldquoa McGuigan protide.&rdquo Designed by medicinal chemist Chris McGuigan in the 1990s, this type of protecting group and its variations are widely used to deliver nucleotide analogs into cells.
&ldquoIt is a brilliant system, because it accomplishes two things,&rdquo Seley-Radtke said. &ldquoNo. 1, an issue with nucleosides is that they&rsquore polar and their phosphates are even more polar.&rdquo Masking the highly negative phosphate groups with esters or amides reduces the molecule&rsquos overall polarity, letting it cross the plasma membrane into cells.
Second, in order to be recognized by polymerases, the analog needs to resemble a normal nucleotide triphosphate&mdashwhich means it needs to be phosphorylated.
&ldquoThe first phosphorylation, either by cellular or viral kinases, is oftentimes very difficult,&rdquo Seley-Radtke said. &ldquoA lot of those kinases are very, very picky in terms of recognition.&rdquo By arriving in the cell with its first phosphate already in tow, remdesivir and related nucleotide analogs skip that rate-limiting step. After the protecting groups are cleaved, the nucleotide analog is a reasonable substrate for later nucletodie kinases.