Articles de revues : « Lin-screen »

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Rocheleau, Christian E., Robyn M. Howard, Alissa P. Goldman, Mandy L. Volk, Laura J. Girard et Meera V. Sundaram. « Alin-45 rafEnhancer Screen Identifieseor-1,eor-2and Unusual Alleles of Ras Pathway Genes inCaenorhabditis elegans ». Genetics 161, n^o 1 (1 mai 2002) : 121–31. http://dx.doi.org/10.1093/genetics/161.1.121.

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AbstractIn Caenorhabditis elegans, the Ras/Raf/MEK/ERK signal transduction pathway controls multiple processes including excretory system development, P12 fate specification, and vulval cell fate specification. To identify positive regulators of Ras signaling, we conducted a genetic screen for mutations that enhance the excretory system and egg-laying defects of hypomorphic lin-45 raf mutants. This screen identified unusual alleles of several known Ras pathway genes, including a mutation removing the second SH3 domain of the sem-5/Grb2 adaptor, a temperature-sensitive mutation in the helical hairpin of let-341/Sos, a gain-of-function mutation affecting a potential phosphorylation site of the lin-1 Ets domain transcription factor, a dominantnegative allele of ksr-1, and hypomorphic alleles of sur-6/PP2A-B, sur-2/Mediator, and lin-25. In addition, this screen identified multiple alleles of two newly identified genes, eor-1 and eor-2, that play a relatively weak role in vulval fate specification but positively regulate Ras signaling during excretory system development and P12 fate specification. The spectrum of identified mutations argues strongly for the specificity of the enhancer screen and for a close involvement of eor-1 and eor-2 in Ras signaling.

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Cherian, Jerrin R., Katherine V. Adams et Lisa N. Petrella. « Wnt Signaling Drives Ectopic Gene Expression and Larval Arrest in the Absence of the Caenorhabditis elegans DREAM Repressor Complex ». G3&#58 ; Genes|Genomes|Genetics 10, n^o 2 (16 décembre 2019) : 863–74. http://dx.doi.org/10.1534/g3.119.400850.

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Establishment and maintenance of proper gene expression is a requirement for normal growth and development. The DREAM complex in Caenorhabditis elegans functions as a transcriptional repressor of germline genes in somatic cells. At 26°, DREAM complex mutants show increased misexpression of germline genes in somatic cells and High Temperature Arrest (HTA) of worms at the first larval stage. To identify transcription factors required for the ectopic expression of germline genes in DREAM complex mutants, we conducted an RNA interference screen against 123 transcription factors capable of binding DREAM target promoter loci for suppression of the HTA phenotype in lin-54 mutants. We found that knock-down of 15 embryonically expressed transcription factors suppress the HTA phenotype in lin-54 mutants. Five of the transcription factors found in the initial screen have associations with Wnt signaling pathways. In a subsequent RNAi suppression screen of Wnt signaling factors we found that knock-down of the non-canonical Wnt/PCP pathway factors vang-1, prkl-1 and fmi-1 in a lin-54 mutant background resulted in strong suppression of the HTA phenotype. Animals mutant for both lin-54 and vang-1 showed almost complete suppression of the HTA phenotype, pgl-1 misexpression, and fertility defects associated with lin-54 single mutants at 26°. We propose a model whereby a set of embryonically expressed transcription factors, and the Wnt/PCP pathway, act opportunistically to activate DREAM complex target genes in somatic cells of DREAM complex mutants at 26°.

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Shimizu, Yoshiaki. « "Chinese Recluse Lin Pu Greeting a Crane" : A Japanese Screen ». Record of the Art Museum, Princeton University 53, n^o 2 (1994) : 2. http://dx.doi.org/10.2307/3774701.

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Abrahante, Juan E., Eric A. Miller et Ann E. Rougvie. « Identification of Heterochronic Mutants in Caenorhabditis elegans : Temporal Misexpression of a Collagen ::Green Fluorescent Protein Fusion Gene ». Genetics 149, n^o 3 (1 juillet 1998) : 1335–51. http://dx.doi.org/10.1093/genetics/149.3.1335.

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Abstract The heterochronic genes lin-4, lin-14, lin-28, and lin-29 specify the timing of lateral hypodermal seam cell terminal differentiation in Caenorhabditis elegans. We devised a screen to identify additional genes involved in this developmental timing mechanism based on identification of mutants that exhibit temporal misexpression from the col-19 promoter, a downstream target of the heterochronic gene pathway. We fused the col-19 promoter to the green fluorescent protein gene (gfp) and demonstrated that hypodermal expression of the fusion gene is adult-specific in wild-type animals and temporally regulated by the heterochronic gene pathway. We generated a transgenic strain in which the col-19::gfp fusion construct is not expressed because of mutation of lin-4, which prevents seam cell terminal differentiation. We have identified and characterized 26 mutations that restore col-19::gfp expression in the lin-4 mutant background. Most of the mutations also restore other aspects of the seam cell terminal differentiation program that are defective in lin-4 mutant animals. Twelve mutations are alleles of three previously identified genes known to be required for proper timing of hypodermal terminal differentiation. Among these are four new alleles of lin-42, a heterochronic gene for which a single allele had been described previously. Two mutations define a new gene, lin-58. When separated from lin-4, the lin-58 mutations cause precocious seam cell terminal differentiation and thus define a new member of the heterochronic gene pathway.

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de la Cova, Claire C., Robert Townley et Iva Greenwald. « Negative feedback by conserved kinases patterns the degradation of Caenorhabditiselegans Raf in vulval fate patterning ». Development 147, n^o 24 (3 novembre 2020) : dev195941. http://dx.doi.org/10.1242/dev.195941.

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ABSTRACTActivation of a canonical EGFR-Ras-Raf-ERK cascade initiates patterning of multipotent vulval precursor cells (VPCs) of Caenorhabditis elegans. We have previously shown that this pathway includes a negative-feedback component in which MPK-1/ERK activity targets the upstream kinase LIN-45/Raf for degradation by the SEL-10/FBXW7 E3 ubiquitin ligase. This regulation requires a Cdc4 phosphodegron (CPD) in LIN-45 that is conserved in BRAF. Here, we identify and characterize the minimal degron that encompasses the CPD and is sufficient for SEL-10-mediated, MPK-1-dependent protein degradation. A targeted screen of conserved protein kinase-encoding genes yielded gsk-3 (an ortholog of human GSK3B) and cdk-2 (a CDK2-related kinase) as required for LIN-45 degron-mediated turnover. Genetic analysis revealed that LIN-45 degradation is blocked at the second larval stage due to cell cycle quiescence, and that relief of this block during the third larval stage relies on activation of CDKs. Additionally, activation of MPK-1 provides spatial pattern to LIN-45 degradation but does not bypass the requirement for gsk-3 and cdk-2. This analysis supports a model whereby MPK-1/ERK, GSK-3/GSK3 and CDK-2/CDK2, along with SEL-10/FBXW7, constitute a regulatory network that exerts spatial and temporal control of LIN-45/Raf degradation during VPC patterning.

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Boxem, Mike, et Sander van den Heuvel. « lin-35 Rb and cki-1 Cip/Kip cooperate in developmental regulation of G1 progression in C. elegans ». Development 128, n^o 21 (1 novembre 2001) : 4349–59. http://dx.doi.org/10.1242/dev.128.21.4349.

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We have investigated the regulation of cell-cycle entry in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, we identified mutations in cyd-1 cyclin D and cdk-4 Cdk4/6. Recent results indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, our data indicate that C. elegans cyd-1 primarily controls G1 progression. To investigate whether cyd-1 and cdk-4 solely act to overcome G1 inhibition by retinoblastoma family members, we constructed double mutants that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of lin-35 Rb, the single Rb-related gene in C. elegans, substantially reduced the DNA replication and cell-division defects in cyd-1 and cdk-4 mutant animals. These results demonstrate that lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for cyd-1 and cdk-4. However, as the suppression by lin-35 Rb is not complete, cyd-1 and cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. We demonstrate that lin-35 Rb and cki-1 Cip/Kip contribute non-overlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of cki-1, but not lin-35, results in precocious entry into S phase. We suggest that a rate limiting role for cki-1 Cip/Kip rather than lin-35 Rb explains the lack of cell-cycle phenotype of lin-35 mutant animals.

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Cohen, Alexandra R., Daniel F. Wood, Shirin M. Marfatia, Zenta Walther, Athar H. Chishti et James Melvin Anderson. « Human CASK/LIN-2 Binds Syndecan-2 and Protein 4.1 and Localizes to the Basolateral Membrane of Epithelial Cells ». Journal of Cell Biology 142, n^o 1 (13 juillet 1998) : 129–38. http://dx.doi.org/10.1083/jcb.142.1.129.

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In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.

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Clark, Denise V., Robert C. Johnsen, Kim S. McKim et David L. Baillie. « Analysis of lethal mutations induced in a mutator strain that activates transposable elements in Caenorhabditis elegans ». Genome 33, n^o 1 (1 février 1990) : 109–14. http://dx.doi.org/10.1139/g90-017.

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A screen was conducted for lethal mutations in the nematode Caenorhabditis elegans in a strain containing the mutator mut-4(st700)I to examine the nature of mutator-induced lethal mutations within two large chromosomal regions comprising a total of 49 map units (linkage group IV (right) and linkage group V (left)). The genetic analysis of 28 lethal mutations has revealed that the mutator locus mut-4(st700)I causes both putative single-gene mutations and deficiencies. We have identified lethal mutations in three different genes, in addition to seven deficiencies. There is a mutational hot spot on linkage group V (left) around the lin-40 locus. Six mutations appear to be alleles of lin-40. In addition, 5 of 7 deficiencies have breakpoints at or very near lin-40. All seven deficiencies delete the left-most known gene on linkage group V (left) and thus appear to delete the tip of the chromosome. This is in contrast to gamma ray and formaldehyde induced deficiencies, which infrequently delete the closest known gene to the tip of a chromosome.Key words: Caenorhabditis elegans, mutator, deficiencies, lethal mutations.

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Hartin, Samantha N., Martin L. Hudson, Curtis Yingling et Brian D. Ackley. « A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis ». PLOS ONE 10, n^o 5 (4 mai 2015) : e0121397. http://dx.doi.org/10.1371/journal.pone.0121397.

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Strutz, Nicole, Hanna Brodowski, Joern Kiselev, Anika Heimann-Steinert et Ursula Müller-Werdan. « App-Based Evaluation of Older People’s Fall Risk Using the mHealth App Lindera Mobility Analysis : Exploratory Study ». JMIR Aging 5, n^o 3 (16 août 2022) : e36872. http://dx.doi.org/10.2196/36872.

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Background Falls and the risk of falling in older people pose a high risk for losing independence. As the risk of falling progresses over time, it is often not adequately diagnosed due to the long intervals between contacts with health care professionals. This leads to the risk of falling being not properly detected until the first fall. App-based software able to screen fall risks of older adults and to monitor the progress and presence of fall risk factors could detect a developing fall risk at an early stage prior to the first fall. As smartphones become more common in the elderly population, this approach is easily available and feasible. Objective The aim of the study is to evaluate the app Lindera Mobility Analysis (LIN). The reference standards determined the risk of falling and validated functional assessments of mobility. Methods The LIN app was utilized in home- and community-dwelling older adults aged 65 years or more. The Berg Balance Scale (BBS), the Tinetti Test (TIN), and the Timed Up & Go Test (TUG) were used as reference standards. In addition to descriptive statistics, data correlation and the comparison of the mean difference of analog measures (reference standards) and digital measures were tested. Spearman rank correlation analysis was performed and Bland-Altman (B-A) plots drawn. Results Data of 42 participants could be obtained (n=25, 59.5%, women). There was a significant correlation between the LIN app and the BBS (r=–0.587, P<.001), TUG (r=0.474, P=.002), and TIN (r=–0.464, P=.002). B-A plots showed only few data points outside the predefined limits of agreement (LOA) when combining functional tests and results of LIN. Conclusions The digital app LIN has the potential to detect the risk of falling in older people. Further steps in establishing the validity of the LIN app should include its clinical applicability. Trial Registration German Clinical Trials Register DRKS00025352; https://tinyurl.com/65awrd6a

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Zhu, Meilin, Tandong Yao, Wei Yang, Baiqing Xu et Xiaojun Wang. « Evaluation of Parameterizations of Incoming Longwave Radiation in the High-Mountain Region of the Tibetan Plateau ». Journal of Applied Meteorology and Climatology 56, n^o 4 (avril 2017) : 833–48. http://dx.doi.org/10.1175/jamc-d-16-0189.1.

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AbstractAccurate evaluations of incoming longwave radiation (Lin) parameterization have practical implications for glacier and river runoff changes in high-mountain regions of the Tibetan Plateau (TP). To identify potential means of accurately predicting spatiotemporal variations in Lin, 13 clear-sky parameterizations combined with 10 cloud corrections for all-sky atmospheric emissivity were evaluated at five sites in high-mountain regions of the TP through temporal and spatial parameter transfer tests. Most locally calibrated parameterizations for clear-sky and all-sky conditions performed well when applied to the calibration site. The best parameterization at five sites is Dilley and O’Brien’s A model combined with Sicart et al.’s A for cloud-correction-incorporated relative humidity. The performance of parameter transferability in time is better than that in space for the same all-sky parameterizations. The performance of parameter transferability in space presents spatial discrepancies. In addition, all all-sky parameterizations show a decrease in performance with increasing altitude regardless of whether the parameters of all-sky parameterizations were recalibrated by local conditions or transferred from other study sites. This may be attributable to the difference between screen-level air temperature and the effective atmospheric boundary layer temperature and to different cloud-base heights. Nevertheless, such worse performance at higher altitudes is likely to change because of terrain, underlying surfaces, and wind systems, among other factors. The study also describes possible spatial characteristics of Lin and its driving factors by reviewing the few studies about Lin for the mountain regions of the TP.

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Simoneaux, D. K., F. A. Fletcher, R. Jurecic, H. G. Shilling, N. T. Van, P. Patel et J. W. Belmont. « The receptor tyrosine kinase-related gene (ryk) demonstrates lineage and stage-specific expression in hematopoietic cells. » Journal of Immunology 154, n^o 3 (1 février 1995) : 1157–66. http://dx.doi.org/10.4049/jimmunol.154.3.1157.

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Abstract We attempted to isolate novel receptor tyrosine kinase, which may play a role in hematopoietic development by screening for expressed sequences with conserved tyrosine kinase catalytic domains. Among the known tyrosine kinases identified in this screen, we found a gene with characteristics of a receptor tyrosine kinase but unusual motifs in the catalytic domain. This gene is identical to ryk described independently by other investigators. Chromosomal fluorescence in situ hybridization localization of human ryk was clarified by using monochromosomal hybrids and placing it as a single locus in 3q22. Although Northern analysis reveals widespread expression in adult mouse tissues, we have found that ryk expression is not ubiquitous. Expression increased in bone marrow cells from mice treated with 5-fluorouracil. Northern analysis on cell lines indicates expression in CD3-, CD4-, CD8- T cells (at a low level), pre-T cells, thymic epithelial cells, and mature myeloid cells, but not myeloid precursors or B cell precursors. Expression analysis with the use of RT-PCR on mouse bone marrow cells separated on the basis of cell surface markers (B220, CD4, CD8, Gr-1, Mac-1) reveals that this receptor is expressed in differentiated cells (Lin+) but is not expressed in the precursor cells (Lin-). Flow cytometric analysis with a monospecific anti-Ryk Ab demonstrates that Ryk+ cells constitute 36.7% and Lin+/Ryk+ cells constitute 33.7% of low density bone marrow cells whereas Ryk+ cells represent only 0.3% of the Lin- population. We conclude that ryk expression is regulated during hematopoietic development by lineage commitment and stage of maturation.

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Nemeth, Michael J., David J. Curtis, Martha R. Kirby, Lisa J. Garrett-Beal, Nancy E. Seidel, Amanda P. Cline et David M. Bodine. « Hmgb3 : an HMG-box family member expressed in primitive hematopoietic cells that inhibits myeloid and B-cell differentiation ». Blood 102, n^o 4 (15 août 2003) : 1298–306. http://dx.doi.org/10.1182/blood-2002-11-3541.

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Abstract Hmgb3 is a member of a family of chromatin-binding proteins that can alter DNA structure to facilitate transcription factor binding. We identified the Hmgb3 cDNA in a subtractive hybridization screen for transcripts that are preferentially expressed in hematopoietic stem cells. We inserted an internal ribosomal entry site–green fluorescence protein cassette into the 3′ untranslated region of the X-linked Hmgb3 locus to identify Hmgb3-expressing cells. In adult mice, Hmgb3 mRNA is detected in bone marrow cells, primitive Lin–, c-kit+, Sca-1+, IL-7Rα– cells, and Ter119+ erythroid cells. We observed that long-term repopulating ability is entirely contained in the subpopulation of Lin–, c-kitHI cells that express Hmgb3. Most common lymphoid and myeloid progenitors express Hmgb3. Introduction of a retrovirus containing the Hmgb3 cDNA into mouse bone marrow stem cells demonstrated that enforced expression of Hmgb3 inhibited B-cell and myeloid differentiation. We conclude that down-regulation of Hmgb3 protein levels is an important step for myeloid and B-cell differentiation.

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Armakola, Maria, et Gary Ruvkun. « Regulation of Caenorhabditis elegans neuronal polarity by heterochronic genes ». Proceedings of the National Academy of Sciences 116, n^o 25 (4 juin 2019) : 12327–36. http://dx.doi.org/10.1073/pnas.1820928116.

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Many neurons display characteristic patterns of synaptic connections that are under genetic control. The Caenorhabditis elegans DA cholinergic motor neurons form synaptic connections only on their dorsal axons. We explored the genetic pathways that specify this polarity by screening for gene inactivations and mutations that disrupt this normal polarity of a DA motorneuron. A RAB-3::GFP fusion protein that is normally localized to presynaptic terminals along the dorsal axon of the DA9 motorneuron was used to screen for gene inactivations that disrupt the DA9 motorneuron polarity. This screen identified heterochronic genes as major regulators of DA neuron presynaptic polarity. In many heterochronic mutants, presynapses of this cholinergic motoneuron are mislocalized to the dendrite at the ventral side: inactivation of the blmp-1 transcription factor gene, the lin-29/Zn finger transcription factor, lin-28/RNA binding protein, and the let-7miRNA gene all disrupt the presynaptic polarity of this DA cholinergic neuron. We also show that the dre-1/F box heterochronic gene functions early in development to control maintenance of polarity at later stages, and that a mutation in the let-7 heterochronic miRNA gene causes dendritic misplacement of RAB-3 presynaptic markers that colocalize with muscle postsynaptic terminals ectopically. We propose that heterochronic genes are components in the UNC-6/Netrin pathway of synaptic polarity of these neurons. These findings highlight the role of heterochronic genes in postmitotic neuronal patterning events.

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Yu, Chun Xuan, et Wei Zhao. « Design and Application of the Multifunctional Digital Automobile Instrument ». Advanced Materials Research 383-390 (novembre 2011) : 2073–76. http://dx.doi.org/10.4028/www.scientific.net/amr.383-390.2073.

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According to the developmental trend of modern automobile instrument, this paper puts forward a multifunctional digital instrument based on the embedded Linux system. The digital instrument panel consists of embedded microprocessor and contact screen, and it could not only receive and process all kinds of driving information from peripheral interface and display the results on the LCD, but also has the function of aided parking. This paper proposes an integrated design solution for the digital instrument. The design selects the TI’s TMS320DM355, which includes LCD and contact screen module, CAN bus module, LIN bus module, video-in and audio-out. The digital instrument can receive real-time data, and also process the data and store it in the RAM and display all vehicle operating parameters in the LCD and realizes real time monitoring in backing up. Based on the experiment, it is proved that the designed system reaches the primary design specifications.

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Maulana, Muhammad Sobri, et Hartono Gunardi. « RISK OF LANGUAGE DELAY IN TODDLERS WITH PROLONGED SCREEN TIME : EVIDENCE BASED CASE REPOR ». JECIES : Journal of Early Childhood Islamic Education Study 1, n^o 1 (30 mars 2020) : 34–48. http://dx.doi.org/10.33853/jecies.v1i1.53.

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Background: Language development of children starts early in infantcy and surges in 2 years of life, updated knowledge about association of language delay with its aggravating risk factor, in this case prolong sreening time, is very important to determine the prognosis of language development in children. Objective: To investigate the association between increased risk of language delay in toddlers with prolonged screen time. Methods: All included studies were collected from Pubmed, Scopus, EBSCO, Clinical Key and Science Direct on February 11th 2020. These articles were then critically appraised using standard Oxford criteria of Evidence Based Medicine prognostic checklist. Result: Two eligible retrospective cohort studies from Lin et al (2014) and Byeon and Hong (2015) are included in this EBCR. Both were calculating the risk of language delay in toddlers between 15-35 months and 24-30 months exposed to screen viewing. Toddlers with more than 2 hours of watching television have higher risk of language delay (Odds Ratio: 3.3 (95%CI 1.5-7.3) and 2.74 (95%CI 1.13-6.65) respectively). Conclusion : The risk of language delay in toddlers is confirmed to be proportionately increased with the increases of screen duration. Maximum language development may be achieved by giving more two-way communication opportunities other than screen viewing.Keywords: Language delay, Toddlers, Screen time. ABSTRAKLatar belakang : Perkembangan bahasa anak-anak dimulai sejak kanak-kanak dan melonjak dalam dua tahun awal kehidupan, pengetahuan terbaru tentang hubungan antara keterlambatan bahasa dengan faktor risiko yang memburuk, dalam hal ini memperpanjang waktu menonton televisi sangat penting untuk menentukan prognosis perkembangan bahasa pada anak-anak. Tujuan : Untuk menyelidiki hubungan antara peningkatan risiko keterlambatan bahasa pada balita dengan waktu menonton televisi yang lama. Metode : Semua studi literatur yang dimasukkan dan dikumpulkan dari Pubmed, Scopus, EBSCO, Clinical Key dan Science direct pada 11 Februari 2020. Jurnal-jurnal ini kemudian di nilai secara appraisal menggunakan kriteria standar Oxford dalam kaidah laporan kasus berbasis bukti. Hasil : Dua studi kohort retrospektif yang memenuhi syarat kaidah laporan kasus berbasis bukti / EBCR yaitu Lin et al (2014) dan Byeon dan Hong (2015) dimasukkan dalam EBCR ini. Keduanya melakukan penelitian dan menghitung risiko keterlambatan bahasa pada balita usia antara 15-35 bulan dan 24-30 bulan yang terpapar menonton televisi dalam waktu lama . Balita yang menonton televise lebih dari dua jam sehari memiliki risiko keterlambatan bahasa yang lebih tinggi masing-masing(Odds Ratio: 3.3 (95%CI 1.5-7.3) dan 2.74 (95%CI 1.13-6.65). Kesimpulan : Risiko keterlambatan bahasa pada balita dipastikan akan meningkat secara proporsional dengan meningkatnya durasi menonton televise. Perkembangan bahasa pada anak lebih maksimal dapat dicapai dengan memberikan lebih banyak waktu untuk berkomunikasi dua arah antara keluarga selain dari menonton televisi.

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Eisenmann, David M., et Stuart K. Kim. « Protruding Vulva Mutants Identify Novel Loci and Wnt Signaling Factors That Function During Caenorhabditis elegans Vulva Development ». Genetics 156, n^o 3 (1 novembre 2000) : 1097–116. http://dx.doi.org/10.1093/genetics/156.3.1097.

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Abstract The Caenorhabditis elegans vulva develops from the progeny of three vulval precursor cells (VPCs) induced to divide and differentiate by a signal from the somatic gonad. Evolutionarily conserved Ras and Notch extracellular signaling pathways are known to function during this process. To identify novel loci acting in vulval development, we carried out a genetic screen for mutants having a protruding-vulva (Pvl) mutant phenotype. Here we report the initial genetic characterization of several novel loci: bar-1, pvl-4, pvl-5, and pvl-6. In addition, on the basis of their Pvl phenotypes, we show that the previously identified genes lin-26, mom-3/mig-14, egl-18, and sem-4 also function during vulval development. Our characterization indicates that (1) pvl-4 and pvl-5 are required for generation/survival of the VPCs; (2) bar-1, mom-3/mig-14, egl-18, and sem-4 play a role in VPC fate specification; (3) lin-26 is required for proper VPC fate execution; and (4) pvl-6 acts during vulval morphogenesis. In addition, two of these genes, bar-1 and mom-3/mig-14, are known to function in processes regulated by Wnt signaling, suggesting that a Wnt signaling pathway is acting during vulval development.

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Degenhardt, Yan Yan, et Saul Silverstein. « Interaction of Zyxin, a Focal Adhesion Protein, with the E6 Protein from Human Papillomavirus Type 6 Results in Its Nuclear Translocation ». Journal of Virology 75, n^o 23 (1 décembre 2001) : 11791–802. http://dx.doi.org/10.1128/jvi.75.23.11791-11802.2001.

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ABSTRACT Zyxin, a focal adhesion molecule, interacts specifically with the E6 protein from human papillomavirus (HPV) type 6 in a yeast two-hybrid screen of a cDNA library prepared from human keratinocytes. Zyxin does not interact significantly with E6 proteins from HPV types 11, 16, or 18. The interaction was confirmed by in vitro and in vivo analyses and it requires the LIM domains (Lin-11, Isl-1, and Mec-3 [G. Freyd, S. K. Kim, and H. R. Horvitz, Nature 344:876–879, 1990]) found at the carboxyl terminus of zyxin. Cotransfection of E6 from HPV (6E6) and zyxin results in the accumulation of zyxin in the nucleus where it can function as a transcriptional activator. 6E6 can also mobilize endogenous zyxin to the nucleus.

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Louvet-Vallée, Sophie, Irina Kolotuev, Benjamin Podbilewicz et Marie-Anne Félix. « Control of Vulval Competence and Centering in the Nematode Oscheius sp. 1 CEW1 ». Genetics 163, n^o 1 (1 janvier 2003) : 133–46. http://dx.doi.org/10.1093/genetics/163.1.133.

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Abstract To compare vulva development mechanisms in the nematode Oscheius sp. 1 to those known in Caenorhabditis elegans, we performed a genetic screen for vulva mutants in Oscheius sp. 1 CEW1. Here we present one large category of mutations that we call cov, which affect the specification of the Pn.p ventral epidermal cells along the antero-posterior axis. The Pn.p cells are numbered from 1 to 12 from anterior to posterior. In wild-type Oscheius sp. 1 CEW1, the P(4-8).p cells are competent to form the vulva and the progeny of P(5-7).p actually form the vulva, with the descendants of P6.p adopting a central vulval fate. Among the 17 mutations (defining 13 genes) that we characterize here, group 1 mutations completely or partially abolish P(4-8).p competence, and this correlates with early fusion of the Pn.p cells to the epidermal syncytium. In this group, we found a putative null mutation in the lin-39 HOM-C homolog, the associated phenotype of which could be weakly mimicked by injection of a morpholino against Osp1-lin-39 in the mother’s germ line. Using cell ablation in a partially penetrant competence mutant, we show that vulval competence is partially controlled by a gonadal signal. Most other mutants found in the screen display phenotypes unknown in C. elegans. Group 2 mutants show a partial penetrance of Pn.p competence loss and an abnormal centering of the vulva on P5.p, suggesting that these two processes are coregulated by the same pathway in Oscheius sp. 1. Group 3 mutants display an enlarged competence group that includes P3.p, thus demonstrating the existence of a specific mechanism inhibiting P3.p competence. Group 4 mutants display an abnormal centering of the vulval pattern on P7.p and suggest that a specific mechanism centers the vulval pattern on a single Pn.p cell.

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Schmitz, Caroline, Parag Kinge et Harald Hutter. « Axon guidance genes identified in a large-scale RNAi screen using the RNAi-hypersensitiveCaenorhabditis elegansstrainnre-1(hd20) lin-15b(hd126) ». Proceedings of the National Academy of Sciences 104, n^o 3 (9 janvier 2007) : 834–39. http://dx.doi.org/10.1073/pnas.0510527104.

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Cui, M., M. A. Allen, A. Larsen, M. MacMorris, M. Han et T. Blumenthal. « Genes involved in pre-mRNA 3'-end formation and transcription termination revealed by a lin-15 operon Muv suppressor screen ». Proceedings of the National Academy of Sciences 105, n^o 43 (22 octobre 2008) : 16665–70. http://dx.doi.org/10.1073/pnas.0807104105.

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Cali, Brian M., Sherry L. Kuchma, Jonathan Latham et Philip Anderson. « smg-7 Is Required for mRNA Surveillance in Caenorhabditis elegans ». Genetics 151, n^o 2 (1 février 1999) : 605–16. http://dx.doi.org/10.1093/genetics/151.2.605.

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Abstract Eukaryotic mRNAs that contain premature stop codons are degraded more rapidly than their wild-type counterparts, a phenomenon termed “nonsense-mediated mRNA decay” (NMD) or “mRNA surveillance.” Functions of six previously described Caenorhabditis elegans genes, smg-1 through smg-6, are required for NMD. Whereas nonsense mutant mRNAs are unstable in smg(+) genetic backgrounds, such mRNAs have normal stability in smg(–) backgrounds. Previous screens for smg mutations have likely not identified all genes involved in NMD, but efforts to identify additional smg genes are limited by the fact that almost 90% of smg mutations identified in genome-wide screens are alleles of smg-1, smg-2, or smg-5. We describe a modified screen for smg mutations that precludes isolating alleles of smg-1, smg-2, and smg-5. Using this screen, we have identified and cloned smg-7, a previously uncharacterized gene that we show is required for NMD. smg-7 is predicted to encode a novel protein that contains an acidic carboxyl terminus and two probable tetratricopeptide repeats. We provide evidence that smg-7 is cotranscribed with the previously characterized gene lin-45 and show that null alleles of smg-7 confer a temperature-sensitive defect in NMD.

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Deng, Yuting, Katherine Leisan Luo, Daniel D. Shaye et Iva Greenwald. « A Screen of the Conserved Kinome for Negative Regulators of LIN-12 Negative Regulatory Region (“NRR”)-Missense Activity in Caenorhabditis elegans ». G3&#58 ; Genes|Genomes|Genetics 9, n^o 11 (13 septembre 2019) : 3567–74. http://dx.doi.org/10.1534/g3.119.400471.

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Chang, Ting-Wei, Chiu-Feng Wang, Hsin-Jye Huang, Iren Wang, Shang-Te Danny Hsu et You-Di Liao. « Key Residues of Outer Membrane Protein OprI Involved in Hexamer Formation and Bacterial Susceptibility to Cationic Antimicrobial Peptides ». Antimicrobial Agents and Chemotherapy 59, n^o 10 (27 juillet 2015) : 6210–22. http://dx.doi.org/10.1128/aac.01406-15.

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ABSTRACTAntimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI fromPseudomonas aeruginosaor its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985–8994, 2010,http://dx.doi.org/10.1074/jbc.M109.078725; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem287:418–428, 2012,http://dx.doi.org/10.1074/jbc.M111.290361). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged withEscherichia colisignal peptide was expressed in a hexamer, which was anchored on the surface ofE. coli, possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as nativeP. aeruginosaOprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials.

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Schoemans, Helene M., Rikkert Snoeckx, Shannon Buckley, Elda Mimiola, Sarah Schouteden et Catherine M. Verfaillie. « RASSF8 Overexpression Expands KLS Cells in Vitro and Blocks Murine Hematopoietic Stem Cells in a Primitive Phenotype. » Blood 112, n^o 11 (16 novembre 2008) : 1409. http://dx.doi.org/10.1182/blood.v112.11.1409.1409.

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Abstract A recent transcriptome analysis of human umbilical cord blood and bone marrow (BM) CD34+CD33−CD38−ckit+Rholo (Rholo) vs. CD34+CD33−CD38−ckit+Rhohi (Rhohi) cells identified the gene RASSF8, to be more highly expressed in the hematopoietic stem cell (HSC) rich compartment (Rholo) versus committed progenitor cells (Rhohi). In a subsequent screen for the function of differentially expressed genes in hematopoiesis, the knockdown of RASSF8 using morpholinos in zebrafish embryos resulted in decreased blood formation and reduced expression of early (scl and gata1) and late (hbae1 and lcp1) hematopoietic markers. To test the function of RASSF8 in mammalian hematopoiesis, we overexpressed RASSF8 cDNA in lineage depleted murine bone marrow cells (Lin−) by retroviral transduction using the MSCV-RASSF8-IRES-GFP vector (rMIG-RASSF8). Sorted transduced GFP+ cells were cultured in serum-free medium (supplemented with SCF, TPO, Flt3L and Il-3) for three to five days. No significant differences were seen in overall cell expansion, cell death (7-AAD staining) or cell proliferation (thymidine incorporation assay and propidium iodide staining) between rMIG-RASSF8 transduced cells and cells transduced with the control vector (rMIG). However, we noted a significant accumulation of the stem cell enriched cKit+Lin−Sca1+ (KLS) population in rMIG-RASSF8 transduced cells (43+/−9%) compared to control cells transduced with rMIG (13+/−8%, p=0.003; n=4) after five days. The total number of colony forming cells (CFCs) produced by 750 rMIG-RASSF8 transduced Lin− cells (24+/−7) was significantly lower than in rMIG transduced cells (54+/−8, p=0.008; n=3). CFCs from rMIG-RASSF8 transduced cells were morphologically smaller and more compact, consistent with the morphology of primitive blast-forming colonies. Competitive repopulation assays using 25 000 rMIG-RASSF8 or rMIG transduced C57/Bl6 CD45.1 Lin- cells versus 100 000 C57/Bl6 CD45.2 mononuclear BM cells were performed to evaluate HSC engraftment potential. Four weeks after transplantation only 1+/−0.96% CD45.1 cells were found in the peripheral blood (PB) of the animals receiving rMIG-RASSF8 transduced cells vs. 23+/−12% CD45.1 cells in animals that had received rMIG transduced cells (p< 0.05; n=3). Likewise, PB analysis at 3 months demonstrated nearly no reconstitution of rMIG-RASSF8 transduced CD45.1 cells (1+/−1.7%) vs 30+/−23% rMIG transduced CD45.1 cells (p=0.02). In conclusion, constitutive overexpression of RASSF8 increases the retention/expansion of KLS cells in vitro, blocks differentiation of progenitors giving rise to CFC and inhibits engraftment of HSC. These studies thus suggest that short term overexpression of RASSF8 might ultimately lead to new methods of HSC expansion.

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Fay, D. S. « fzr-1 and lin-35/Rb function redundantly to control cell proliferation in C. elegans as revealed by a nonbiased synthetic screen ». Genes & ; Development 16, n^o 4 (15 février 2002) : 503–17. http://dx.doi.org/10.1101/gad.952302.

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Mehta, Nehal, Paula M. Loria et Oliver Hobert. « A Genetic Screen for Neurite Outgrowth Mutants in Caenorhabditis elegans Reveals a New Function for the F-box Ubiquitin Ligase Component LIN-23 ». Genetics 166, n^o 3 (mars 2004) : 1253–67. http://dx.doi.org/10.1534/genetics.166.3.1253.

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Cramer, Kimberly, Margaret Nieborowska-Skorska, Artur Slupianek, Tomasz Sliwinski, David Irvine, Mhairi Copland et Tomasz Skorski. « RAD52-Dependent Synthetic Lethality Eradicates Leukemia Stem Cells ». Blood 120, n^o 21 (16 novembre 2012) : 3. http://dx.doi.org/10.1182/blood.v120.21.3.3.

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Abstract Abstract 3 We showed that imatinib-naive and imatinib-treated BCR-ABL1 kinase-positive chronic myeloid leukemia in chronic phase (CML-CP) Lin−CD34+CD38− stem cells (LSCs) and Lin−CD34+CD38+ leukemia progenitor cells (LPCs) accumulate high numbers of the reactive oxygen species (ROS)-induced DNA double-strand breaks (DSBs) which, if not repaired, are lethal (Nieborowska-Skorska et al., Blood, 2012). Genome-wide microarray screen indicated that LSCs overexpress numerous genes responsible for homologous recombination repair (HRR) of DSBs. Direct targeting of RAD51, a key element in HRR, by small molecule inhibitor exerted anti-CML effect, but normal cells were also affected, indicating that RAD51 is not a preferable target. In normal cells HRR depends primarily on BRCA1/BRCA2-RAD51 pathway, while RAD52-RAD51 pathway may serve as a back-up. However BRCA1 protein is downregulated in CML, and RAD51(F259V) mutant (RAD52 binding-deficient) induced apoptosis and reduced cell growth in BCR-ABL1 –dependent manner, thus highlighting the potential role of alternative RAD52-driven HRR in CML. The absence of RAD52 (Rad52−/−) inhibited BCR-ABL1 –mediated cell cycle progression and clonogenic activity, induced apoptosis, reduced Lin−Kit+Sca1+CD34−Flt3− long-term LSCs and Lin−Kit+Sca1+CD34+Flt3− short-term LSCs, and abrogated leukemogenesis in murine model of CML. At the same time RAD52 was expendable in normal cells. In addition, RAD52 was essential to prevent accumulation of ROS-induced lethal DSBs in BCR-ABL1 –positive murine LSCs. Thus it appears that RAD52 is necessary to repair numerous ROS-induced DSBs in LSCs to promote leukemogenesis. BCR-ABL1 kinase interacts with and phosphorylates RAD52 on Y104, but phosphorylation-deficient RAD52(Y104F) mutant did not exert any negative effect on BCR-ABL1 kinase-driven leukemogenesis. This observation suggests that RAD52 activity is preserved in imatinib-treated cells. On the other hand, DNA binding-deficient RAD52(F79A) and RAD52(K102A) mutants (disrupt DNA binding domain I and II, respectively) inhibited clonogenic potential of Lin−CD34+ CML-CP cells and BCR-ABL1 –positive Lin−Kit+Sca1+ murine LSCs, but not normal counterparts. Therefore, RAD52-mediated DNA binding activity appears to be essential for BCR-ABL1 kinase-driven leukemogenesis, but not normal hematopoiesis. RAD52 DNA I binding groove containing F79 forms a pocket/niche. Peptide aptamer containing amino acid residues surrounding RAD52 F79, but not that with F79A substitution, inhibited RAD52 foci, RAD52-dependent RAD51 foci and HRR activity, resulting in elevation of the number of lethal DSBs and abrogation of clonogenic activity of BCR-ABL1-positive murine leukemia cells. Aptamer F79 reduced the growth of LSCs and LPCs from CML-CP and more aggressive CML-accelerated phase (CML-AP), depleted quiescent LSCs by attrition and eradicated BCR-ABL1 leukemia from SCID mice. Moreover, aptamer F79 enhanced the anti-CML effects of imatinib. The effect of aptamer F79 in BCR-ABL1 leukemia cells depended on downregulation of BRCA1 implicating “synthetic lethality”. At the same time the aptamer did not exert any measurable negative effects on normal cells and tissues. In conclusion, we postulate that CML-CP/AP cells are “addicted” to RAD52 and that targeting of DNA binding domain of RAD52 may induce “synthetic lethality” to eliminate proliferating LSCs/LPCs and to deplete quiescent LSCs. Moreover, anti-RAD52 F79 aptamer exerted anti-tumor activity in acute myeloid leukemia primary cells, and in cell lines derived from carcinomas of breast, ovary and pancreas displaying BRCA1 and/or BRCA2 deficiency. Thus, BRCA1/BRCA2ness-driven “addiction” to RAD52 may be selectively targeted to achieve “synthetic lethality” in wide-range of tumors while being harmless to normal cells. Disclosures: Copland: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

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Coraggio, Francesca, Ringo Püschel, Alisha Marti et Peter Meister. « Polycomb and Notch signaling regulate cell proliferation potential duringCaenorhabditis eleganslife cycle ». Life Science Alliance 2, n^o 1 (26 décembre 2018) : e201800170. http://dx.doi.org/10.26508/lsa.201800170.

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Stable cell fate is an essential feature for multicellular organisms in which individual cells achieve specialized functions.Caenorhabditis elegansis a great model to analyze the determinants of cell fate stability because of its invariant lineage. We present a tractable cell fate challenge system that uses the induction of fate-specifying transcription factors. We show that wild-type differentiated animals are highly resistant to fate challenge. Removal of heterochromatin marks showed marked differences: the absence of histone 3 lysine 9 methylation (H3K9) has no effect on fate stability, whereas Polycomb homologmes-2mutants lacking H3K27 methylation terminally arrest larval development upon fate challenge. Unexpectedly, the arrest correlated with widespread cell proliferation rather than transdifferentiation. Using a candidate RNAi larval arrest-rescue screen, we show that the LIN-12Notchpathway is essential for hyperplasia induction. Moreover, Notch signaling appears downstream of food-sensing pathways, as dauers and first larval stage diapause animals are resistant to fate challenge. Our results demonstrate an equilibrium between proliferation and differentiation regulated by Polycomb and Notch signaling in the soma during the nematode life cycle.

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Rotelli, Michael D., Anna M. Bolling, Andrew W. Killion, Abraham J. Weinberg, Michael J. Dixon et Brian R. Calvi. « An RNAi Screen for Genes Required for Growth of Drosophila Wing Tissue ». G3&#58 ; Genes|Genomes|Genetics 9, n^o 10 (6 août 2019) : 3087–100. http://dx.doi.org/10.1534/g3.119.400581.

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Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila. The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or βNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development.

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He, Qiuping, Mengzhi Hong, Jincan He, Weixin Chen, Meng Zhao et Wei Zhao. « Isoform-specific involvement of Brpf1 in expansion of adult hematopoietic stem and progenitor cells ». Journal of Molecular Cell Biology 12, n^o 5 (30 septembre 2019) : 359–71. http://dx.doi.org/10.1093/jmcb/mjz092.

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Abstract Bromodomain-containing proteins are known readers of histone acetylation that regulate chromatin structure and transcription. Although the functions of bromodomain-containing proteins in development, homeostasis, and disease states have been well studied, their role in self-renewal of hematopoietic stem and progenitor cells (HSPCs) remains poorly understood. Here, we performed a chemical screen using nine bromodomain inhibitors and found that the bromodomain and PHD finger-containing protein 1 (Brpf1) inhibitor OF-1 enhanced the expansion of Lin−Sca-1+c-Kit+ HSPCs ex vivo without skewing their lineage differentiation potential. Importantly, our results also revealed distinct functions of Brpf1 isoforms in HSPCs. Brpf1b promoted the expansion of HSPCs. By contrast, Brpf1a is the most abundant isoform in adult HSPCs but enhanced HSPC quiescence and decreased the HSPC expansion. Furthermore, inhibition of Brpf1a by OF-1 promoted histone acetylation and chromatin accessibility leading to increased expression of self-renewal-related genes (e.g. Mn1). The phenotypes produced by OF-1 treatment can be rescued by suppression of Mn1 in HSPCs. Our findings demonstrate that this novel bromodomain inhibitor OF-1 can promote the clinical application of HSPCs in transplantation.

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Stefanovska, Bojana, Kevin Lin, Benjamin Troness, Chad Myers et Reuben Harris. « Abstract P2-17-01 : Targeted CRISPR screen to identify synthetic lethal combinations between APOBEC3B and DNA repair ». Cancer Research 83, n^o 5_Supplement (1 mars 2023) : P2–17–01—P2–17–01. http://dx.doi.org/10.1158/1538-7445.sabcs22-p2-17-01.

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Abstract APOBEC-catalyzed deamination of cytosine bases is the largest enzymatic and second largest overall source of mutation in cancer. One member from the APOBEC family of enzymes, APOBEC3B (A3B) is overexpressed and dysregulated in many different cancer types. In addition to hallmark C-to-T transitions and C-to-G transversions, APOBEC-catalyzed uracil lesions can be processed into single- and double-strand DNA breaks. Therefore, A3B-positive tumors are under continual stress to repair DNA breaks and may be vulnerable to DNA repair inhibition. Previous results from the Harris lab have identified UNG2, DNA uracil glycosylase 2 (UNG2), initiator of the base excision repair pathway, as synthetic lethal pair with A3B. The genetic disruption of UNG2 combined with high expression of A3B, causes cell death. This provides the rational to hypothesize that also other DNA repair proteins could be putative synthetic lethal pairs with A3B, when its expression and activity are high. To test this hypothesis, CRISPR guide RNA library targeting 237 DNA damage repair and response genes was used in doxycycline-inducible TREX-293-A3Bi-eGFP cell line. Cells expressing or not A3B were harvested for DNA extraction and sequencing at different time points. Comparison of guide RNA abundance between doxycycline and H2O treated cells revealed the dropout guides that disrupt genes and create putative synthetic lethal combinations with A3B. The screen design and overall results will be presented Citation Format: Bojana Stefanovska, Kevin Lin, Benjamin Troness, Chad Myers, Reuben Harris. Targeted CRISPR screen to identify synthetic lethal combinations between APOBEC3B and DNA repair [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-01.

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Walker, Denise S., Sung Ly, Nicholas J. D. Gower et Howard A. Baylis. « IRI-1, a LIN-15B Homologue, Interacts with Inositol-1,4,5-Triphosphate Receptors and Regulates Gonadogenesis, Defecation, and Pharyngeal Pumping in Caenorhabditis elegans ». Molecular Biology of the Cell 15, n^o 7 (juillet 2004) : 3073–82. http://dx.doi.org/10.1091/mbc.e04-01-0039.

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Inositol-1,4,5-triphosphate receptors (IP3Rs) are ligand-gated Ca2+ channels that control Ca2+ release from intracellular stores. They are central to a wide range of cellular responses. IP3Rs in Caenorhabditis elegans are encoded by a single gene, itr-1, and are widely expressed. Signaling through IP3 and IP3Rs is important in ovulation, control of the defecation cycle, modulation of pharyngeal pumping rate, and embryogenesis. To further elucidate the molecular basis of the diversity of IP3R function, we used a yeast two-hybrid screen to search for proteins that interact with ITR-1. We identified an interaction between ITR-1 and IRI-1, a previously uncharacterized protein with homology to LIN-15B. Iri-1 is widely expressed, and its expression overlaps significantly with that of itr-1. In agreement with this observation, iri-1 functions in known itr-1-mediated processes, namely, upregulation of pharyngeal pumping in response to food and control of the defecation cycle. Knockdown of iri-1 in an itr-1 loss-of-function mutant potentiates some of these effects and sheds light on the signaling pathways that control pharyngeal pumping rate. Knockdown of iri-1 expression also results in a sterile, evl phenotype, as a consequence of failures in early Z1/Z4 lineage divisions, such that gonadogenesis is severely disrupted.

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Jiang, Xiaoyan, Kyi Min Saw, Allen Eaves et Connie Eaves. « Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency. » Blood 106, n^o 11 (16 novembre 2005) : 438. http://dx.doi.org/10.1182/blood.v106.11.438.438.

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Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

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Ting, Stephen, Eric Deneault, Melanie Frechette, Jalila Chagraoui et Guy Sauvageau. « A Functional Screen Identifies Polarity Genes Implicated in HSC Fate ». Blood 112, n^o 11 (16 novembre 2008) : 732. http://dx.doi.org/10.1182/blood.v112.11.732.732.

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Abstract The molecular details governing self-renewal in tissue stem cells of the invertebrate systems of Drosophila Melanogaster and C. Elegans have been instructive for equivalent tissues in vertebrates. In the aforementioned invertebrates, an integral group of genes involved in cell polarity seem able to intrinsically act as or affect cell fate determinants (CFDs) during the process of stem cell asymmetric cell division (ACD). On this premise, we focused on potential polarity genes that may act as CFD during HSC self-renewal. 72 CFD candidates were chosen from a literature review that addressed mechanisms of ACD. Gene expression profiles were performed on both highly purified Long Term Repopulating-HSC populations and primary Leukemia Stem Cells. A significant number of these candidates were highly and differentially expressed. The highest ranking 60% of candidates (42 of the initial 72 genes) was then chosen for a functional in vitro to in vivo over-expression screen. The underlying theory of this screen is based on the ability of Hoxb4-induced HSCs, as compared to control vector-induced HSCs, to expand during a short in vitro culture period, together with their ability to provide significant long-term reconstitution upon transplantation after this in vitro expansion. Therefore, a positive candidate would be one that has a Hoxb4-like expansion effect on HSCs. In brief, using a 96 well plate format, 1500 CD150+48-Lin-Ly5.1+ donor derived HSCs were infected independently with each candidate, together with negative (vector alone) and positive (Hoxb4 and Nup98-Hoxa10 fusion) controls, for a total of 12 days and equal proportions of HSCs were transplanted after 5 and 12 days of in vitro culture into recipient Ly5.2+ mice. The read out measurement was donor Ly5.1+ peripheral blood reconstitution performed at monthly intervals for 5 months. At day 5 transplantations, 12 of the 42 genes had donor reconstitution above the empty vector control at 16 weeks. Of these 12 genes, only 4 retained positive long-term transplant donor reconstitution after the extra week of infection to 12 days. These 4 genes were: Ap2a2, Gpsm2, Tmod1 and Kif3a. Of these, the first 2 genes are robust candidates, having been replicated in 4 independent experiments. Interestingly, both these CFD candidates, Ap2a2 (as part of the endocytic machinery that interacts with membrane receptors) and Gpsm2 (as a G-protein signaling modulator that also influences mitotic spindle orientation) potentially provide mechanisms that allow the HSC to communicate with the niche. Ap2a2 induced HSCs in particular are able to reconstitute to levels beyond and equivalent to Hoxb4 and Nup98-HoxA10-induction, respectively. Oligoclonality (ruling out insertional mutagenesis) and multipotency from donor-derived Ly5-1+ HSCs in recipients at 20 plus weeks post-transplantation has also been performed. Endogenous Ap2a2 is localized predominantly asymmetrically in purified LTR-HSCs, as opposed to a predominant symmetrical distribution in E14 fetal liver HSCs. Initial live cell microscopy of LTR-HSCs infected with Ap2a2 fluorescent fusion proteins confirms the asymmetrical distribution, and further mechanistic insights should follow with prolonged video microscopy.

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Sanjiv, Kumar, José Manuel Calderón-Montaño, Therese M. Pham, Tom Erkers, Viktoriia Tsuber, Ingrid Almlöf, Andreas Höglund et al. « MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia ». Cancer Research 81, n^o 22 (30 septembre 2021) : 5733–44. http://dx.doi.org/10.1158/0008-5472.can-21-0061.

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Abstract Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin−CD34+CD38−), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. Significance: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

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Kerssens, Chantal. « Aging and Gaming : The Science and Promise of Technology-Based Leisure Activities and Interventions ». Innovation in Aging 4, Supplement_1 (1 décembre 2020) : 558. http://dx.doi.org/10.1093/geroni/igaa057.1835.

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Abstract Much research has focused on technology to support older adults in basic and instrumental activities of daily living. Much less is known about technology supports of hobbies and leisure later in life. Physical, cognitive and social activities potentially delay the onset and progression of disease, including dementia. Older adults are interested in digital games, applications (apps) and social technologies, and will use technology provided their needs, preferences and goals are met. Moreover, video games can be designed to promote satisfying social experiences between players of differing capabilities. More work, however, is needed to understand older adults’ interactions and engagement with game-based interventions. This symposium presents cutting-edge research findings and design recommendations for technology-based leisure activities and interventions in older adults. Yow et al. present data from a large, touch-screen dual language intervention program with cognitive training tools aimed at slowing down the rate of cognitive decline in older adults with dementia. Boot et al. present longitudinal data from the Center for Research and Education on Aging and Technology Enhancement (CREATE) with a focus on leisure and videogames; Freed et al. present older adults’ attitudes and experiences with an exercise videogame (exergame). Lin et al. discuss the early effects of an exergame involving real-world physical activity on activity, social contact and stress levels in dementia caregivers. Kerssens et al. discuss the creation and testing of an adapted, accessible version of beloved board games for people with mild cognitive impairment (MCI) and a care partner without MCI. Technology and Aging Interest Group Sponsored Symposium.

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Kao, Li-Ling. « ESG-Based Performance Assessment of the Operation and Management of Industrial Parks in Taiwan ». Sustainability 15, n^o 2 (11 janvier 2023) : 1424. http://dx.doi.org/10.3390/su15021424.

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The development of industrial parks plays an important role in the economic development of developed and developing countries, but it has recently been affected by globalization and the rise of environmental protection awareness, as 2050 net-zero carbon emissions are being pursued by the world’s major economies. To make their development more sustainable, this study evaluates the operational management performance of industrial parks in Taiwan from the perspective of ESG, to inform future industrial development strategies and policy research. First, 61 industrial parks managed by the Taiwanese Ministry of Economic Affairs (MOEA) were selected and underwent a fuzzy Delphi expert questionnaire to screen the ESG-oriented performance indicators; performance was evaluated through the data envelopment analysis (DEA) undesirable outputs model and the window analysis method. The results indicate that the New Taipei Industrial Park performed best in terms of ESG, followed by the Feng-Shan and Anping industrial parks, with the worst performance from the Mei-Lun, Yun-Lin Island, and Hu-Pin industrial parks. Regarding factors affecting the performance of operation management, a Mann–Whitney U test showed that the northern industrial parks performed significantly better than those in the eastern region, those in the municipalities significantly outperformed the nonmunicipalities, and the industrial parks with more clustered industries and those in areas with convenient transportation performed substantially better. Finally, this study summarizes the important issues facing Taiwan’s industrial parks, and it makes policy recommendations, including promoting ESG sustainable development objectives in their operation and management, as well as increasing the investment of government resources and the clustering of industries and transportation.

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Makowski, Michal, Paweł Piątek et Mateusz Grynkiewicz. « Projection of holographic images in volumetric fluorescent fluids for near-eye displays ». Photonics Letters of Poland 11, n^o 4 (31 décembre 2019) : 99. http://dx.doi.org/10.4302/plp.v11i4.936.

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The optical setup for holographic projection on the scatterings in fluorescent liquids is presented. Such media can be used as volumetric screens for near-eye holographic displays, solving the problem of speckle noise and very small exit pupils in existing setups. Three different oils (canola, olive and engine oil) with 532 nm laser and tonic water with 405 nm laser are used for projecting holographic fields, the quality of such images is investigated. The laser wavelength is cut out from acquisition on a camera and only filtered fluorescent light is observed. The best and brightest results are obtained with engine oil. Full Text: PDF ReferencesX. Li, C. P. Chen, H. Gao, et al. "Video-Rate Holographic Display Using Azo-Dye-Doped Liquid Crystal", Journal of display technology 10(6), 438-443 (2014). CrossRef X. Li, Z. Song, F. Li, X. Dong, W. Liu, "79‐3: Video‐rate Holographic Display in ZnSe layer‐assisted Quantum Dot Doped Liquid Crystal with High‐photorefractive Sensitivity", SID Symposium Digest of Technical Papers. Vol. 48. No. 1. 2017, CrossRef Sasaki, Takeo, et al. "Real-time dynamic hologram in photorefractive ferroelectric liquid crystal with two-beam coupling gain coefficient of over 800 cm–1 and response time of 8 ms", Applied Physics Letters 6(2) (2013) CrossRef N. Tsutsumi, K. Kinashi, A. Nomura, W. Sasaki, "Quickly Updatable Hologram Images Using Poly(N-vinyl Carbazole) (PVCz) Photorefractive Polymer Composite", Materials 5.8: 1477-1486 (2012) CrossRef M. Makowski, "Simple holographic projection in color", et al. Optics express 20.22: 25130-25136 (2012) CrossRef A. Yagi, M. Imura, Y, Kuroda, O. Oshiro, "360-degree fog projection interactive display", SIGGRAPH Asia 2011 Emerging Technologies. ACM, (2011) CrossRef C.H. Hsu, K. L. Hua, W. H. Cheng. "Omni-Tube: a low-cost portable omnidirectional interactive 3D display", SIGGRAPH Asia 2012 Posters. ACM, (2012) CrossRef Z. Zeng, H. Zheng, X. Lu, H. Gao, Y. Yu, "Dynamic holographic three-dimensional projection based on liquid crystal spatial light modulator and cylindrical fog screen", Opt Rev (2015) 22: 853 CrossRef I. Rakkolainen, "Feasible mid-air virtual reality with the immaterial projection screen technology", 3DTV-Conference, Tampere (2010) CrossRef S. Yanfeng, et al. "A multi-plane optical see-through holographic three-dimensional display for augmented reality applications", Optik 157: 190-196 (2018) CrossRef G. Li, D. Lee, Y. Jeong, J. Cho, B. Lee, "Holographic display for see-through augmented reality using mirror-lens holographic optical element", Opt. Lett. 41(11), 2486-2489 (2016) CrossRef C. L. Lin, Y. Z. Su, M. W. Hung, K. C. Huang "Augmented reality system", Proc. SPIE 7798, Applications of Digital Image Processing XXXIII, 779826 (2010) CrossRef A. Maimone, A. Georgiou, J. S. Kollin, "Holographic near-eye displays for virtual and augmented reality", ACM Trans. Graph. 36, 4, 1-16 (2017) CrossRef M. Quinten, Optical properties of nanoparticle systems: Mie and beyond (John Wiley & Sons 2010). CrossRef J.-W. Liaw, S.-W. Tsai, H.-H. Lin, T.-C. Yen, B.-R. Chen, "Wavelength-dependent Faraday–Tyndall effect on laser-induced microbubble in gold colloid", Journal of Quantitative Spectroscopy and Radiative Transfer 113(17), 2234-2242 (2012), CrossRef T. Mu et al. "Classification of edible oils using 532 nm laser-induced fluorescence combined with support vector machine", Anal. Methods 5, 6960 (2013) CrossRef T. Mu et al. "Classification of Motor Oil Using Laser-Induced Fluorescence and Phosphorescence", Analytical Letters 49:8, 1233-1239 (2015) CrossRef V. Rostampour, M. J. Lynch, "Quantitative Techniques To Discriminate Petroleum Oils Using LED-induced Fluorescence", WIT Transactions on Ecology and the Environment 95, 265 262 (2006) CrossRef F. Wyrowski and O. Bryngdahl, "Iterative Fourier-transform algorithm applied to computer holography", Opt. Soc. Am. A 5(7), 1058-1065 (1988) CrossRef

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Lin, Chang-Ching, Tsung-Cheng Chang, Alberto Servetto, Kyung-min Lee, He Zhang, Yunguan Wang, Dan Ye et al. « Abstract P5-17-09 : A genome-wide CRISPR screen identifies PRMT5 as a novel therapeutic target in ER+/RB1-deficient breast cancer ». Cancer Research 82, n^o 4_Supplement (15 février 2022) : P5–17–09—P5–17–09. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-17-09.

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Abstract Background: CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with advanced estrogen receptor-positive (ER+) breast cancer. However, this benefit is transient as virtually all these tumors eventually develop drug resistance and recur. Clinical studies have reported an association of RB1 loss-of-function genomic alterations with acquired resistance to CDK4/6i. Given the enrichment of RB1 alterations post CDK4/6i treatment, ER+/RB1-deficient breast cancer will become a rising patient population in need of discovery of novel treatment strategies. In this study, we sought to identify actionable vulnerabilities for this refractory breast cancer subtype using a genome-wide CRISPR screen. Methods: RB1 was knocked out in ER+ MCF-7 and T47D breast cancer cells using CRISPR-Cas9; complete gene knockout was confirmed by PCR-based genotyping, Sanger sequencing, and immunoblot analysis. Isogenic RB1 knockout (RBKO) and wild-type (WT) T47D cells were used for the genome-wide CRISPR screen. MAGeCKFlute was used to identify differentially essential genes in T47D RBKO vs WT cells; Gene Ontology (GO) analysis was used to prioritize hits. MCF-7 and T47D RBKO cells were used for validating and studying the function of the identified genes. Results: Knockout of RB1 in MCF-7 and T47D cells increased IC50 of abemaciclib, palbociclib, and ribociclib 10-200 fold compared to WT cells. RNA-seq analysis showed upregulation of E2F target gene expression in RBKO vs WT cells. The CRISPR screen revealed that CCND1 and CDK4 lost their essentiality in T47D RBKO cells, suggesting that loss of RB1 uncouples the CDK4/Cyclin D1 complex from E2F-regulated transcription. GO analysis of the top 50 differentially essential hits of RBKO vs WT cells showed an enrichment of protein arginine methyltransferase activity, primarily PRMT5, which post-translationally mono-methylates and symmetrically di-methylates protein arginine. In agreement with this finding, PRMT5 knockout by three individual sgRNAs resulted in more potent growth inhibition of MCF-7 and T47D RBKO cells than WT cells. Further, transfection of PRMT5 siRNA or treatment with the PRMT5 small molecule inhibitor GSK3326595 - currently in clinical trials - resulted in G1 arrest of MCF-7 and T47D RBKO cells as assayed by propidium iodide staining but did not induce caspase 3/7 or PARP cleavage (apoptosis). RNA-seq of PRMT5 siRNA vs control siRNA in MCF-7 and T47D RBKO cells exhibited significant downregulation of E2F Hallmark gene signature, further suggesting PRMT5 inhibition as a strategy to suppress E2F-regulated gene expression when cells lose Rb. The CRISPR screen also revealed that transcription factors that drive ER signaling, such as FOXA1, GATA3, MYC, SPDEF, and ESR1 (the gene encoding ERα), were commonly essential in both T47D WT and RBKO cells. Estrogen deprivation or treatment with fulvestrant inhibited estrogen responsive element (ERE) luciferase reporter activity, expression of putative E2F target genes, and proliferation of both WT and RBKO cells, suggesting that ER+ cells still rely on ERα irrespective of RB1 status. Treatment of MCF-7 and T47D RBKO cells with fulvestrant and GSK3326595 resulted in more potent growth inhibition than each drug alone, suggesting a novel approach to treat ER+/RB1-deficient breast cancer. We are currently testing the antitumor activity of fulvestrant plus GSK3326595 against RBKO xenografts as well as the requirement of arginine methyltransferase activity associated with PRMT5 for growth of ER+/RB1-deficient breast cancer cells. Conclusion: PRMT5 is essential for proliferation of ER+/RB1-deficient breast cancer cells. Targeting PRMT5 in combination with anti-estrogens is a novel and testable strategy to suppress E2F-regulated cell cycle progression of this CDK4/6 inhibitor-resistant breast cancer subtype. Citation Format: Chang-Ching Lin, Tsung-Cheng Chang, Alberto Servetto, Kyung-min Lee, He Zhang, Yunguan Wang, Dan Ye, Sumanta Chatterjee, Dhivya R Sudhan, Hiroaki Akamatsu, Yang Xie, Joshua T Mendell, Ariella B Hanker, Carlos L Arteaga. A genome-wide CRISPR screen identifies PRMT5 as a novel therapeutic target in ER+/RB1-deficient breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-09.

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Leli, Nektaria Maria, Souvik Dey, Lauren Brady, Carlo Salas Salinas, Jerome Lin, Giorgos Skoufos, Ioannis Verginadis, Artemis Chatzigeorgiou et Constantinos Koumenis. « Abstract 144 : Survivin, a novel mediator of the UPR identified by a functional genome wide CRISPR/Cas9 based knock out screen ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 144. http://dx.doi.org/10.1158/1538-7445.am2022-144.

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Abstract Tumor growth and metastasis involve a complex relationship with the tissue microenvironment. A proliferating tumor encounters several stresses from the microenvironment like hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells can co-opt existing cytoprotective mechanisms, such as the Unfolded Protein Response (UPR). The UPR involves transcriptional and translational activation of signaling pathways designed to relieve cellular stress and attenuate cell death; although, in the case of unresolved or chronic ER stress UPR can promote cell death. However, the mechanisms by which the UPR influences cell fate in the presence of ER stress are poorly understood. To address this, we have used a functional CRISPR-based genetic knockout screen, to determine novel regulators of UPR and the mechanisms involved to control cellular fate following chronic ER stress. We delivered a lentiviral genome-wide CRISPR-Cas9 knockout library to two cell lines and identified sgRNAs which are over-represented (pro-apoptotic genes), or underrepresented (pro-survival genes) following ER stress induced by thapsigargin and tunicamycin. One of the highest-ranking gene candidates was Survivin/BIRC5, a protein which is overexpressed in tumor cells compared to normal tissues. Survivin was first identified as an inhibitor of apoptosis but also participates in the regulation of the cell cycle. Survivin held a high position in the negative ranking scale of all screens suggesting a possible prosurvival role in response to ER stress. In agreement, our results show that genetic knock down or chemical inhibition of Survivin led to sensitization of cells to ER stress. Moreover, both in vitro an in vivo data support that PERK (a key sensor of the third arm of the UPR cascade) is responsible for this sensitization. Interestingly, we observed that ER stress in turn leads to downregulation of Survivin in a dose and time dependent fashion, both in the level of protein but also RNA, employing a variety of mechanisms like protein translation, degradation and RNA synthesis. Foreseeably, PERK knock down reverses the aforementioned effect supporting its role as a key participant in the interplay between Survivin and ER stress. Though the balance between those two is still to be understood our results demonstrate that Survivin holds a novel function as an ER stress regulator but also targeting of PERK and Survivin in a clinical setting could reveal new windows in therapeutic intervention in malignancy. Citation Format: Nektaria Maria Leli, Souvik Dey, Lauren Brady, Carlo Salas Salinas, Jerome Lin, Giorgos Skoufos, Ioannis Verginadis, Artemis Chatzigeorgiou, Constantinos Koumenis. Survivin, a novel mediator of the UPR identified by a functional genome wide CRISPR/Cas9 based knock out screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 144.

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Velu, Chinavenmeni S., Anil G. Jegga, Vivek Kaimal, Bruce Aronow et H. Leighton Grimes. « miR-21 Is a Transcriptional Target of Growth Factor Independent -1 ; Implications for Transcriptional Regulation of microRNA. » Blood 108, n^o 11 (16 novembre 2006) : 1178. http://dx.doi.org/10.1182/blood.v108.11.1178.1178.

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Abstract Hematopoiesis is a tightly regulated multistage process, wherein pluripotent self-renewing stem cells gives rise to all blood cell lineages. Growth factor independent -1 (Gfi1) is a transcription factor that relies on corepressor proteins such as G9a to repress transcription of target genes. Gfi1 is required for hematopoietic stem cell self-renewal and subsequent granulocytic lineage development. Recent studies have identified microRNAs (miRs) as 21–23 nucleotide non-coding RNA that regulate gene expression. It is currently unclear which microRNAs control hematopoiesis. The deregulated Gfi1 null environment provides a unique setting to screen for miRNA that may control hematopoiesis. Therefore, we performed microRNA gene expression array analyses on RNA extracted from total bone marrow cells of Gfi1 null and wild type littermate mice. Our results demonstrate that several miR genes are differentially expressed in Gfi1 knock-out mice; however, we have focused further analyses on miR-21, which was consistently upregulated in Gfi1 null cells. Thus, miR-21 provides a potential direct target for Gfi1. Moreover, miR-21 was previously shown to regulate apoptosis in neuronal cell lines. As demonstrated by both expression array and Taqman, the relative expression of the mature miR-21 in Gfi1 null bone marrow cells is more than 7 fold higher than wild type Gfi1 littermate bone marrow. Because loss of Gfi1 alters hematopoiesis, we normalized the cells under analysis by lineage marker depletion. Still, in comparison to similar Lin-populations from wild type littermates, the expression of miR-21 was consistently higher in Gfi1 null Lin-bone marrow cells. We next attempted to determine whether miR-21 is directly regulated by Gfi1; however, bioinformatic analyses of microRNA regulatory sequences are currently underdeveloped. We therefore adopted a systematic gene-centric pipeline approach and adapted a web-accessible database resource termed Genome TraFac (http://genometrafac.cchmc.org) to microRNA orthologs that are conserved between human and murine genomes. The new “miRBase” analyzed for the occurrence of conserved individual cis-elements and compositionally similar cis-culsters to identify validated transcription factor targets within promoter and distal genomic regulatory regions of microRNA genes. Interestingly, many microRNA genes contain conserved regulatory sequences within transcribed precursor sequences, not in the predicted promoter regions. Within the miR-21 gene, two putative Gfi1 binding sites were identified within transcribed precursor sequences. To determine the accuracy of our bioinformatic analyses, we performed chromatin immunoprecipitation (ChIP) with anti-Gfi1 and an anti-G9A antisera. Our data reveal Gfi1, along with its co-factor G9A, is bound to the predicted region (but not control regions lacking predicted Gfi1 binding sites). Notably, electrophoretic mobility shift assay (EMSA) indicated that in vitro transcribed and translated Gfi1 binds strongly to one of the two putative Gfi1 binding sites in the miR-21 gene. These data suggest that the level of miR-21 is regulated directly by Gfi1, and that microRNA genes are commonly regulated by conserved cis-elements within transcribed precursor sequences. The role of miR-21 in controlling differentiation/proliferation in hematopoietic stem cells is currently under investigation.

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Jiang, Yajian, Xiangguo Shi, Kenneth Eagle, Charles Y. Lin et Daisuke Nakada. « Integrating Enhancer Profiling and CRISPR Dropout Screen Revealed Selenoprotein Synthesis Pathway As a New Vulnerability in AML ». Blood 134, Supplement_1 (13 novembre 2019) : 639. http://dx.doi.org/10.1182/blood-2019-126904.

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Alterations of the epigenetic landscape and transcription are hallmarks of acute myeloid leukemia (AML) that drive leukemogenic gene expression and therefore can be exploited for therapeutic intervention. To look for such targets that harbor both an altered epigenetic feature and are genetically essential for AML cells, we performed a multi-database analysis integrating pan-cancer super enhancer landscapes with whole genome CRISPR dropout screens. Among the top targets, we discovered SEPHS2. An enhancer was present upstream of the gene marked by H3K27ac and bound by leukemogenic transcription factors including MYB, Pu.1 and RUNX1. In addition, AML cells with SEPHS2 deletion significantly dropped out in a genome wide CRISPR screen. This gene encodes a critical enzyme in the underappreciated selenoprotein synthesis pathway which was highly upregulated in TCGA AML patients compared to control blood cells from healthy individuals. Collectively, our initial bioinformatic analysis suggested that the selenoprotein synthesis pathway is a new vulnerability in AML. To test the functional requirement of the selenoprotein synthesis pathway in AML and other cells, we performed CRISPR mediated deletion of three key genes in the selenoprotein synthesis pathway, SEPHS2, SEPSECS and EEFSEC. The human AML cell lines (MOLM13, THP1 and Kasumi1), murine AML cells transformed by MLL-AF9 and human AML PDX cells all depicted a significant dependency on these genes, while proliferation of normal cord blood cells and myeloma cells (U266B1) was almost not affected. We then transplanted these cells into recipients. Deletion of SEPHS2, SEPSECS or EEFSEC significantly ameliorated AML progression, indicated by decreased AML burden and extended survival. Since selenoproteins including GPX1 and GPX4 are known antioxidants, we hypothesized that perturbing the selenoprotein synthesis pathway disrupted the redox state in AML cells. We found that deletion of SEPHS2, SEPSECS or EEFSEC elevated ROS in AML cells as demonstrated by Cell-Rox or DCF-DA staining. Western blotting revealed significantly downregulated GPX4 level and upregulated DNA damage marker γ-H2AX. Moreover, the defective proliferation was partially rescued by adding antioxidant TEMPOL. These results suggested selenoprotein synthesis pathway produced key antioxidants to balance the proper redox state and was required for AML cell proliferation. A major source of selenium is diet. Therefore, we hypothesized that consuming selenium low diet could suppress AML. We compared the survival of AML bearing mice on selenium proficient and deficient diet. The selenium deficient diet significantly extended survival, lowered GPX4 level and increased ROS in AML cells. Interestingly, normal mouse on selenium deficient diet for a 3-months period did not develop any abnormalities in CBC or bone marrow hematopoiesis. This suggests selenium deficient diet could be clinically applicable without significant side effects. Altogether, the integration of a pan-cancer enhancer landscape study with CRISPR dropout gene screen offered a powerful tool to dissect cancer targets that possessed unique enhancer features and genetic essentiality. The analysis yielded SEPHS2 and its selenoprotein synthesis pathway to be a new vulnerability in AML. The underappreciated selenoprotein synthesis pathway was key to produce antioxidant selenoproteins such as GPX1 and GPX4 to maintain a proper redox state in AML. Deleting the genes or removing selenium from diet could perturb the pathway and ameliorate the AML disease. Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

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Jäger, Roland, Markus Muellner, Florian Grebien, Thorsten Klampfl, Ashot Harutyunyan, Damla Olcaydu, Tiina Berg, Sebastian Nijman et Robert Kralovics. « In Vivo Screening for Tumor Suppressors In Hematological Malignancies Using Barcode RNAi ». Blood 116, n^o 21 (19 novembre 2010) : 998. http://dx.doi.org/10.1182/blood.v116.21.998.998.

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Abstract Abstract 998 Chromosomal deletions are frequent cytogenetic defects in hematological malignancies. Common deleted regions (CDRs) defined by the minimal physical overlap of all deletion events are thought to harbor tumor suppressors relevant for pathogenesis. Haploinsufficiency is likely to be a common consequence of chromosomal deletions affecting the function of tumor suppressors within the deleted locus. The use of RNA interference (RNAi) offers a unique opportunity to mimick haploinsufficiency by partial knock-down of the gene transcripts. Since deletions are usually large containing a high number of candidate genes, we aimed to develop a screening method targeting several candidates at once and assaying for tumor suppressor features in a pool of knock-downs. As a model for our approach we selected a CDR on chromosome 20q frequently found clonal in myeloid diseases. We established a screen for knock-downs causing cytokine hypersensitivity. This CDR (physical position chr20:38.7- 42.2) spans 3.5Mb and contains 16 genes (MAFB-JPH2). We cloned 3 short hairpin RNAs (shRNAs) for each mouse homologue of the 16 target genes into the pLKO.2 lentiviral vector. The 48 shRNA constructs were “bar-coded” with different, unique 24bp DNA bar-codes, that can be identified and quantified using the microbead-based xMAP technology (Luminex). We lentivirally delivered the constructs independently into the erythropoietin (Epo) dependent murine cell line Baf3/EpoR, pooled the individual knock-downs in equal amounts and assayed for cytokine hypersensitivity based proliferation advantage under stringent Epo concentrations. Applying a scoring system based on the relative increase/decrease of the individual bar-codes in the pool over time, we could identify the knock-down of topoisomerase 1 (Top1) to induce Epo-concentration dependent outgrowth. Two different Top1 shRNA constructs succeeded in consistently mediating proliferative advantage in three biological replicates. We set up a validation experiment pooling Top1 knock-down cells with control cells (bar-coded, no shRNA) in a 1:1 ratio and could observe significant dominance of the Top1 knock-down cells establishing after 10–15 days in culture and increasing over time (p<0.0001). Knock-down efficiency of both successful constructs measured by qPCR and was consistently between 30–60% of control cell mRNA expression. Based on the convincing cell line data we set up an in vivo validation in a competitive repopulation mouse model. We established a protocol for lentiviral transduction of murine lineage depleted (lin-) bone marrow progenitor cells. Transplanting in a 1:1 ratio of Top1 knock-down and control progenitors into three lethally irradiated mice, we surprisingly observed an outcome opposite to the cell line results. Top1 knock-down progenitors showed a clear disadvantage in repopulation capacity, being underrepresented in the peripheral blood at week 3 post-transplantation and furthermore fully outcompeted by the control cells around week 15 post-transplantation. Based on the unreliability of cell line models in our setup we repeated the screen with the full library (48 shRNAs) in vivo. Two different constructs targeting phospholipase C gamma 1 (Plcg1) had the top score in 3 out of 5 transplanted mice, resulting in an impressive cumulative score. Showing equal representation with the other knock-downs (approximately 2%) in the donor pool, Plcg1 knock-down cells represented up to 13% of the peripheral blood cells at week 3 post- transplant, increasing to up to 29% at week 7 post-transplant. Our results suggest that cell lines often might not be the proper model for studying growth regulation. Further, our in vivo screen revealed Plcg1 as a promising candidate with a tumor suppressor function in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Kamminga, Leonie M., Leonid Bystrykh, Sita Houwer, Jose Douma, Ellen Weersing, Bert Dontje et Gerald de Haan. « Bypassing Cellular Senescence by Ezh2. » Blood 104, n^o 11 (16 novembre 2004) : 2776. http://dx.doi.org/10.1182/blood.v104.11.2776.2776.

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Abstract In this study mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) were used to identify genes involved in the process of cellular aging. MEFs are able to undergo a distinct number of population doublings, after which they enter a state of irreversible growth arrest termed replicative senescence. Previous studies of others and us have shown that aging is regulated by an intrinsic genetic program. In order to identify candidate genes involved in senescence we assessed gene expression profiles of proliferating and senescent MEFs. Interestingly, a significant number of genes that was higher expressed in proliferating vs. senescent MEFs, are involved in epigenetic regulation of gene transcription by modulating histone methylation and deacetylation. The top candidate gene was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG), involved in histone methylation and deacetylation and known to function as a negative regulator of gene transcription. It has been reported that Ezh2 is essential during development, since Ezh2-deficient mice die early during embryonic development. Ezh2 expression was rapidly downregulated during senescence in primary MEFs, but was readily detectable in spontaneously transformed cells. Retroviral overexpression of Ezh2 in primary MEFs resulted in bypassing the senescence program. In addition, overexpression of Ezh2 in spontaneously transformed MEFs increased proliferation rates. We found that Ezh2 is highly expressed in purified Lin−Sca-1+c-kit+ (LSK) HSCs. In agreement with MEF studies, terminal differentiation of stem cells in vitro resulted in rapid downregulation of Ezh2 expression. Furthermore, overexpression of Ezh2 in purified HSCs results in increased proliferation rate in vitro. Moreover, HSCs transduced with Ezh2 were enriched for CFU-GM activity. Ongoing studies will address whether overexpression of Ezh2 in stem cells will affect their self-renewal potential in vivo. In an extensive genetic screen we identified genes that correlate with Ezh2 expression in stem cells, potential targets or partners of Ezh2, among others Top2a, MKi67, Pcna, and Eed. These data were deposited in an on-line resource (www.WebQTL.org), to uncover genetic networks that are involved in regulating the process of aging.

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Yu, Jinpu, et Wenwen Zhang. « 532 SOCS3 deficiency blocked autophagy-dependent myeloid differentiation of early-stage myeloid-derived suppressor cells via the miR-155/C/EBPß/Wnt axis ». Journal for ImmunoTherapy of Cancer 8, Suppl 3 (novembre 2020) : A568. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0532.

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BackgroundEarly-stage myeloid-derived suppressor cells (eMDSCs) are a newly defined subset of myeloid-derived suppressor cells (MDSCs) that accumulate densely in tumors and potently promote tumor growth and metastasis by suppressing antitumor immune responses in vitro and in vivo. We previously identified a subset of eMDSCs in human breast cancer with a characteristic phenotype of Lin-HLA-DR-CD33+. We also found that SOCS3 deficiency and sustained activation of the JAK/STAT signaling pathway are critical molecular events coordinating the differentiation of eMDSCs, although the distinct molecular regulation has not been fully elucidated.MethodsHerein, we genetically constructed conditional SOCS3 knockout mice with SOCS3 deficiency specifically in the myeloid linage (SOCS3MyeKO). We analyzed the number of eMDSCs in SOCS3MyeKO mice (eMDSCsSOCS3KO). To explore which pathways participated in dysfunctional eMDSC differentiation, we performed whole-genome RNA sequencing and miRNA microarray on CD11b+Gr-1+ cells, eMDSCsfl/fl and eMDSCsSOCS3KO to screen the potential regulatory ceRNA network in eMDSCsSOCS3KO. CD11b+Gr-1+ cells isolated from SOCS3fl/fl mouse spleens were used as mature myeloid cell controls. Furthermore, we applied a specific miR-155 antagonist and the autophagy agonist rapamycin to suppress tumor growth and eMDSC infiltration.ResultsThe transcriptome results and corresponding intervention experiment revealed that the differentiation block in eMDSCsSOCS3KO was caused by SOCS3 deficiency-mediated limited autophagy activation in an AMPK-independent manner. The results of miRNA microarray and RNA sequencing demonstrated that miR-155 overexpression and Wnt/ß-catenin pathway activation were involved in the SOCS3 knockout-mediated myeloid differentiation block and autophagy repression. Further experiments revealed that miR-155 was induced by activation of the STAT3/NK-?B pathway upon SOCS3 deficiency, which consequently activated the Wnt/ß-catenin pathway via targeting C/EBPß. Furthermore, applying a specific miR-155 antagonist or the autophagy agonist rapamycin efficiently suppressed tumor growth and eMDSC infiltration in vivo.ConclusionsOverall, these findings indicated that SOCS3 deficiency blocked autophagy-dependent myeloid differentiation of e-MDSCs via the miR-155/C/EBPß/Wnt axis, and thus targeted therapy against this pathway could be a potential therapeutic target in breast cancer.

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TAY, Enoch S. E., Kim L. GUVEN, Nanthakumar SUBRAMANIAM, Patsie POLLY, Laura L. ISSA, Peter W. GUNNING et Edna C. HARDEMAN. « Regulation of alternative splicing of Gtf2ird1 and its impact on slow muscle promoter activity ». Biochemical Journal 374, n^o 2 (1 septembre 2003) : 359–67. http://dx.doi.org/10.1042/bj20030189.

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A human MusTRD [muscle TFII-I repeat domain (RD)-containing protein] isoform was originally identified in a yeast one-hybrid screen as a protein that binds the slow fibre-specific enhancer of the muscle gene troponin I slow [O'Mahoney, Guven, Lin, Joya, Robinson, Wade and Hardeman (1998) Mol. Cell. Biol. 18, 6641–6652]. MusTRD shares homology with the general transcription factor TFII-I by the presence of diagnostic I-RDs [Roy (2001) Gene 274, 1–13]. The human gene encoding MusTRD, GTF2IRD1 (WBSCR11/GTF3), was subsequently located on chromosome 7q11.23, a region deleted in the neurodegenerative disease, Williams–Beuren Syndrome [Osborne, Campbell, Daradich, Scherer, Tsui, Franke, Peoples, Francke, Voit, Kramer et al. (1999) Genomics 57, 279–284; Franke, Peoples and Francke (1999) Cytogenet. Cell. Genet. 86, 296–304; Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737–747]. The haploinsufficiency of MusTRD has been implicated in the myopathic aspect of this disease, which manifests itself in symptoms such as lowered resistance to fatigue, kyphoscoliosis, an abnormal gait and joint contractures [Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737–747]. Here, we report the identification of 11 isoforms of MusTRD in mouse skeletal muscles. These isoforms were isolated from a mouse skeletal muscle cDNA library and reverse transcription–PCR on RNA from various adult and embryonic muscles. The variability in these isoforms arises from alternative splicing of a combination of four cassettes and two mutually exclusive exons, all in the 3′ region of the primary transcript of Gtf2ird1, the homologous mouse gene. The expression of some of these isoforms is differentially regulated spatially, suggesting individual regulation of the expression of these isoforms. Co-transfection studies in C2C12 muscle cell cultures reveal that isoforms differentially regulate muscle fibre-type-specific promoters. This indicates that the presence of different domains of MusTRD influences the activity exerted by this molecule on multiple promoters active in skeletal muscle.

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Chanal, Philippe, et Michel Labouesse. « A Screen for Genetic Loci Required for Hypodermal Cell and Glial-Like Cell Development During Caenorhabditis elegans Embryogenesis ». Genetics 146, n^o 1 (1 mai 1997) : 207–26. http://dx.doi.org/10.1093/genetics/146.1.207.

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The Caenorhabditis elegans lin-26 gene is expressed in all nonneuronal ectodermal cells. To identify genes required to specify the fates of ectodermal cells, we have conducted screens designed to identify loci whose zygotic function would be required for normal lin-26 expression. First, we examined 90 deficiencies covering 75% of the genome; second, we examined the progeny of 3600 genomes after EMS mutagenesis. We identified six loci that appear to be required for normal lin-26 expression. We argue that the deficiency eDf19 deletes a gene involved in specifying hypodermal cell fates. The genes emb-29 (previously known) and ale-1 (newly found) could be involved in a cell cycle function and/or in specifylng the fates of some precursors within different lineages that generate hypodermal cells and nonectodermal cells. We argue that the overlapping deficiencies qDf7, qDf8 and qDf9 delete a gene required to limit the number of nonneuronal ectodermal cells. We suggest that the deficiencies ozDf2, itDf2 and nDf42 delete genes required, directly or indirectly, to repress lin-26 expression in cells that normally do not express lin-26. We discuss the implications of these findings concerning the generation of the ectoderm.

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Crook, Zachary R., Emily J. Girard, Gregory P. Sevilla, Mi-Youn Brusniak, Peter B. Rupert, Della J. Friend, Mesfin M. Gewe et al. « Abstract 1043 : Advances in cystine-dense peptide (CDP) screening and therapeutic applications ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 1043. http://dx.doi.org/10.1158/1538-7445.am2022-1043.

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Abstract Cystine-dense peptides (CDPs) are a class of drug-like miniproteins that marry many of the advantages of biologics (high affinity and specificity) and small molecule therapeutics (high tissue permeability and low immunogenicity). The beneficial properties of CDPs, and miniproteins in general, have driven interest in therapeutic applications. However, CDP diversity is vast from every clade of life, and properly interrogating “CDP space” requires specialized screening and modeling tools. With this in mind, we have created an optimized mammalian surface display platform to screen for CDPs of clinical interest using libraries of structurally-diverse native scaffolds optimized for stability. These native CDPs can be structurally modeled, which we did in determining the structures of over 4200 native CDPs. This modeling permits further selection in silico as well as targeted mutagenesis for favorable target-binding capabilities. Hits from these screens are routinely matured to sub-nM affinity. These CDPs can play numerous roles in a drug design pipeline, from an independent drug candidate to a delivery agent for tissue-targeting to a module in a polyspecific biologic. Recent novel CDP candidates have shown promise in immune-oncology space as part of a bispecific T-cell engager targeting PD-L1, where a single 2-week treatment was capable of eliminating subcutaneous PC3 prostate cancer xenograft tumors in 27/30 mice. Besides bispecifics, future directions for the platform include exploring targeted protein degradation. Additionally, we are expanding upon our previous work on CDPs to explore CNS or tumor delivery of therapeutic cargo. The versatility of CDPs and novel screening tools to rapidly identify and mature candidates of interest can facilitate rapid advancement of CDP therapeutics to address difficult targets in oncology. Citation Format: Zachary R. Crook, Emily J. Girard, Gregory P. Sevilla, Mi-Youn Brusniak, Peter B. Rupert, Della J. Friend, Mesfin M. Gewe, Midori Clarke, Ida Lin, Raymond Ruff, Doan Phi, Ashok Bandaranayake, Colin E. Correnti, Andrew J. Mhyre, Natalie W. Nairn, Roland K. Strong, James M. Olson. Advances in cystine-dense peptide (CDP) screening and therapeutic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1043.

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Carlston, Colleen, Robin Weinmann, Natalia Stec, Simona Abbatemarco, Francoise Schwager, Jing Wang, Huiwu Ouyang, Collin Y. Ewald, Monica Gotta et Christopher M. Hammell. « PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans ». PLOS Genetics 17, n^o 11 (22 novembre 2021) : e1009599. http://dx.doi.org/10.1371/journal.pgen.1009599.

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microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential “prion-like” domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59’s localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation.

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