The best opinions, comments and analysis from The Telegraph. Proof of Funding for Visa: U.S. immigration law requires that international applicants, if admitted, show documented evidence that sufficient funds to cover all tuition, fees, transportation, and living expenses are available for the first year of their studies at UCLA. We're collecting information about the 2022 e-bike rebate program to better understand the impact the rebate had on the community and the possibility of extending the program beyond 2023. [18] The project is sponsored by the U.S. State Department. All the lessons below are free to read on the website. Interestingly, a few lncRNAs have recently been shown to negatively regulate nearby genes through different mechanisms, including the heart transcription factor gene Hand2 (refs. Statistical significance was determined by 2 tests for n=6 wild-type and mutant livers (V1) and n=3 wild-type and mutant livers (V2). Single-molecule fluorescence in situ hybridization was performed as described62. Long non-coding RNA HNF1A-AS1 promotes hepatocellular carcinoma cell proliferation by repressing NKD1 and P21 expression. For differential expression, all cell clusters with >250 cells were grouped in a single cluster. MIN6 cells60 were maintained in DMEM, 4.5gl1 glucose, 15% heat-inactivated FBS, 50M -mercaptoethanol and 50gml1 gentamicin. Oligonucleotides are provided in Supplementary Table 5. Subsequent analyses were carried out with Seurat (version 3.0.1)73 or scVI-tools (version 0.11.0)74. J. g,h, Haster was decreased in Hnf1a/ islets (g; n=4 Hnf1a/ and n=5 Hnf1a+/+ mice) and liver (h; n=4 mice per genotype). This, the Centre said, was omitted from the edited MEMRI video. [75] Supported by Georgetown colleagues, Barakat denied the claim,[76] which Roshwald had based on MEMRI's translation of Barakat's essay.[75]. Chromatin was diluted four times with ChIP Dilution Buffer (0.75% Triton X-100, 0.1% sodium deoxycholate, 140mM NaCl, 50mM HEPES pH8, 1mM EDTA and 1 protease inhibitor cocktail) and 5% was used as input. [24] The project disseminates jihadi-associated social media content and propaganda released by various Islamic State media companies. Out of 33 enhancer-like regions in 1megabase surrounding HNF1A, only E showed significant HNF1A-dependent changes (Extended Data Fig. lincRNAs act in the circuitry controlling pluripotency and differentiation. A maximum of 12 undergraduate units may be counted toward the degree. For US Federal Government officers or employees acting as part of their official duties, the terms are as stated in accordance with our licence terms. Sharing your work in progress will help you determine what you need to concentrate on next. Statistical significance was determined by two-tailed t-test. If applying after March 2, please check with the Financial Aid Office for available funding opportunities. Cas9 from pLV-hIP-Cas9-BSD was replaced by dCas9 using Gibson Assembly to generate pLV-hIP-dCas9-BSD. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Extended Data Fig. & Pritchard, J. K. WASP: allele-specific software for robust molecular quantitative trait locus discovery. c, Colocalization of single molecule fluorescent in situ hybridization signals for HASTER (exonic probes) and HNF1A (intronic probes for HNF1A) in human EndoC-H3 cells. Male mice with germline mutations (Haster/) were born at Mendelian rates and showed no overt manifestations, but also showed diabetes, glucose intolerance and hypoinsulinaemia (Fig. The launch. Spatial Relationship - Distance between objects on stage; one body in relation to another, to a group, or to the architecture. The Middle East Media Research Institute (MEMRI; officially the "Middle East Media and Research Institute") is a nonprofit press monitoring and analysis organization co-founded by former Israeli military intelligence officer Yigal Carmon and Israeli-American political scientist Meyrav Wurmser.Headquartered in Washington, D.C., MEMRI publishes and distributes free Seurat objects were exported as loom using as.loom of the loomR (version 0.2.0.1) library. Register for Banff Viewpoints and take our latest survey. RPM, reads per million reads. Annu. and JavaScript. Questions, please contact the editor at editor@sitnews.us or call 617-9696. e,f, Doxycycline-induced HNF1A overexpression in HASTER-deleted EndoC-H3 cells (n=4) showing (e) normalized HNF1A mRNA levels and (f) HNF1A promoter viewpoint (triangle) UMI-4C contacts. Methods 15, 10531058 (2018). a, Chromatin and RNA maps in mouse (top) and human islets (bottom). PubMed Authors of improvement reports are strongly encouraged to consult the. ISSN 1476-4679 (online) Tbp-normalized mean s.d. Students should expect to be in-person for some, but not all, course components. Libraries were sequenced on a HiSeq 2500 using 2125bp reads or a NovaSeq S4 using 2150bp reads. Diabetes 50, 24722480 (2001). 25, 22272241 (2011). 4j,k). 5f,g). We generated lentiviral vectors with a human insulin promoter driving the expression of Cas9 or dCas9, in addition to a U6-driven single guide RNA (sgRNA) (pLV-hIP-Cas9-BSD (plasmid 183230; Addgene) and pLV-hIP-dCas9-BSD (plasmid 183231; Addgene)). HNF1Alow cells were less abundant than expected from immunostainings, possibly due to a known propensity of Hnf1a knockout cells to dissociate during islet isolation. 2gi). Solid arrowhead: HNF1Ahigh acinar cell. Schultz, M. D. et al. Viral particles were concentrated with 1:3vol/vol of Lenti-X Concentrator (Clontech) at 4C overnight. Studies of homozygous Hnf1a null mutant mice have shown that HNF1A is essential for differentiated cell programs in various organs, whereas human HNF1A haploinsufficiency causes diabetes due to selective abnormalities in pancreatic cells, indicating that the gene dosage sensitivity of HNF1A is cell specific18,21,22,23,24,25,26. 101, 22152222 (1998). Analysis of published datasets showed that HASTER is activated during the early stages of hESC-derived pancreatic differentiation39 (Fig. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. Hnf1a was quantified by strain-specific qPCR and normalized to Tbp (n=4 mice per genotype). Biol. These studies have uncovered a cis-regulatory element that senses HNF1A concentrations and feeds back on HNF1A to ensure appropriate cell-specific expression levels (Fig. 21, 32343243 (2001). International relations (IR) (sometimes referred to as international studies and international affairs) is the scientific study of interactions between sovereign states. They articles should conform to the same guidelines outlined above for Original research. Mol. Cebola, I. Deletion of regulatory elements with all-in-one CRISPRCas9 vectors. Methods 12, 10611063 (2015). Dr Karmi is here sitting with us, and he's very familiar with the jurisprudence. 5d). Similar results were observed in germline Haster mutant mice (Extended Data Fig. It further notes, that MEMRI's founders, Wurmser and Carmon, "are both hardline pro-Israel ideologues aligned with Israel's Likud party". NOTE: UCLA is participating in the U/RTA auditions in New York (Jan 18-21), Chicago (Jan 26-29), and San Francisco on (Feb 1-2). Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. g, Immunofluorescence for HNF1A, PDX1 (a pancreatic and duodenal marker) and glucagon in Haster/ and control E11.5 embryos, showing low heterogeneous HNF1A in pancreatic but not gut progenitors. Alternatively, they should contribute to developing approaches to measuring quality of care in routine practice. for 1h, with an additional hour after the addition of 75l 20% Triton X-100. For UMI-4C comparisons, Pvalues were calculated by 2 test using umi4c. Nat. To quantify tissue specificity, for each gene and tissue we calculated a Z-score that represents the deviation of expression in that tissue relative to the average from all tissues. volume24,pages 15281540 (2022)Cite this article. Our studies exemplify a genetic defect in a mammalian lncRNA promoter that causes an in vivo physiological phenotype. HASTER-dependent negative feedback was essential to prevent HNF1A binding to inappropriate genomic regions. Assoc. [82], In 2002 Brit Hume of Fox News said, "These people tell you what's going on in pulpits and in the state-controlled TV. Nat. Middle East Media and Research Institute Inc. Form 990: Return of Organization Exempt from Income Tax, "Narratives of terrorism and security: 'accurate' translations, suspicious frames", Israeli Web site Debka.com at center of New York dirty bomb tip, "Officials: Israel outsources monitoring of Palestinian media after IDF lapse", "Group exposing the drumbeat of Islamic State's propaganda machine", "State Dept. In ad, the data are presented as TBP-normalized relative expression (meanss.d.) Box plots show medians and interquartile ranges; whiskers, 1.5 times the interquartile ranges. 1 for information on transcriptional start sites. EndoC-H3 cells59 were maintained on a 2gml1 fibronectin- and 1% extracellular matrix-coated plate in Dulbeccos modified Eagle medium (DMEM) low glucose (1gl1), sodium pyruvate (Thermo Fisher Scientific), 2% BSA Fraction V (Roche), 1% heat-inactivated foetal bovine serum (FBS; Labtech), 2mM l-glutamine, 5.5gml1 human transferrin, 1mM sodium pyruvate 10mM nicotinamide, 6.7ngml1 sodium selenite, 50M -mercaptoethanol, 100Uml1 penicillin and 100gml1 streptomycin. Cells were dissociated with TrypLE and seeded in AggreWell 400 plates (34425; Stem Cell Technologies) on day 10. We previously had recommended to authors that they contact us before writing any potential manuscript for this series in order to vet the topic. 10b). A highly physicalized approach to time-honored theater conservatory courses in voice, speech and movement, including, but not limited to, recurring training in the Suzuki and Viewpoints methods. UMI counts were normalized for library size and log transformed. acting audition dates and locations in 2021. Our studies expose a cis-regulatory element that is unlike classic enhancers or silencers, it stabilizes the transcription of its target gene and ensures the fidelity of a cell-specific transcription factor program. By the time of entrance, Theater graduate applicants must: Please visit the FAQ page for additional questions, call 310-206-8441, or e-mail theatergrad@tft.ucla.edu. They may cover any aspect, from clinical or therapeutic intervention, to promotion, to prevention. as the degree, Theater as the major, and Acting as the emphasis. Bioinformatics 35, 20842092 (2019). The Master of Fine Arts degree is awarded on the basis of successful completion of the required courses and critical evaluation of student work. These results suggested that the HASTER promoter functions as a HNF1A-sensing platform that drives HASTER transcription in accordance with HNF1A concentrations. Nat. Briefly, ice-cold collagenase P solution (1mgml1 in Hanks balanced salt solution (HBSS) buffer; Roche) was injected through the main duct. In a broader sense, it concerns all activities between statessuch as war, diplomacy, trade, and foreign policyas well as relations with and among other international actors, such as intergovernmental For visualization, MACS2 bdgcmp was used to generate the local Poisson test log10[Pvalue] bedGraphs. Although the function of most lncRNAs has not been explored, some lncRNAs are known to regulate gene transcription3,4. Differentially expressed genes (adjusted P0.05) are highlighted in red and total numbers are indicated (n=5 mice per genotype). The problem with red, amber, green: the need to avoid distraction by random variation in organisational performance measures. Wang, K. C. et al. Cell 75, 921932 (2019). 6a). acting program at UCLA TFT: Students are required to enroll in a minimum of twelve units per quarter. For more information about this requirement, visit https://grad.ucla.edu/admissions/required-academic-records/. h, Top HOMER de novo motifs for the different categories of HNF1A peak. Thus, HASTER negatively regulates HNF1A in mouse and human hepatic cells. 1a). (n=5 independent experiments). [34], According to its website, MEMRI provides translations and analysis of Arabic, Farsi, Dari, Urdu, Pashto, and Turkish media. Experience everything from thought-provoking viewpoints to diverse cultures to lifelong friendships and life-changing opportunities. We show that HASTERs dual function emanates from a 320-bp promoter DNA sequence and does not require transcription. Subcellular fractionation of EndoC-H3 human cells showed that HASTER transcripts were associated with chromatin, and single-molecule fluorescence in situ hybridization showed that HASTER transcripts were exclusively present in one or two nuclear foci that co-localized with HNF1A nascent transcripts (Fig. The following previously published data were re-analysed: mouse liver ChIP-seq for CTCF (GSE29184; ref. Deletion #1 and #2 were generated with independent pairs of sgRNAs, HASTER+/+ clones were transfected with sgRNAs targeting the AAVS1 locus. Enrichment analysis of de novo transcription factor motifs was also performed with the findMotifsGenome.pl command on consensus peaks defined by DiffBind, for lengths of 8, 10 and 12bp on the masked mm10 genome. Required courses will be scheduled to permit completion of the program within a three-year period. Huang, P. et al. HASTER was strongly downregulated in pancreatic islets and liver from homozygous Hnf1a null mutant mice and in HNF1A-deficient EndoC-H3 human cells (Fig. Alignment statistics are listed in Supplementary Table 2. c, GSEA showing upregulation in HasterpKO cells of genes downregulated in Hnf1a KO islets. [15][16][17] The project also aims to provide a platform for those sources to expand their reach. NOTE: Community college transcripts are not necessary, as the coursework will be reflected on the undergraduate transcript. : not analyzed because deletions were performed at a later time point. You can also explore these lessons if you dont feel ready to perform in front of others or if youre just not sure if you want to act yet. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Human, chicken, Xenopus tropicalis and zebrafish liver RNA-seq reads were aligned on the GCRh37 (hg19), galGal5, XenTro9 and GRCz11 (danRer11) genomes, respectively. In contrast, HasterLKO H3K4me3 showed spreading from the Hnf1a promoter to an intronic E enhancer region (Fig. Cho, S. W. et al. Interaction frequencies were measured at a 5kb region centered on E highlighted with a green shade. 5a). Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 2a) (n=5 independent differentiations). It is often that a performer finds one of the elements comes naturally, and perhaps uses that one element they really understand to access the other elements, which they must work to become more familiar with. Because the wild-type Haster allele was not able to activate the wild-type Hnf1a, which was located on the alternative chromosome, this shows that Haster positively regulates Hnf1a in cis in islet cells. f, Relative proportions of cells present in the HASTERpKO-enriched HNF1Alow cell cluster. MIN6-SAM cells were subsequently transduced with sgRNA expressing vector lenti sgRNA-(MS2)-zeo (61427; Addgene). 4e,f). 9d). However, the Da'wah Centre charged that by omitting the context of Hussein's statements, MEMRI had distorted the actual intent of the speech. In keeping with this notion, HNF1A neo-binding sites did not show accessible chromatin in normal liver (Fig. Results show that HNF1A silencing correlates with time of Haster knockout, with higher silencing frequency after early deletion (Haster germline KO and HasterpKO models). Hnf1a+/ mice have been described24. For more information about this requirement, visit https://grad.ucla.edu/admissions/english-requirements/, Academic Records: Although the Application for Graduate Admission enables applicants to upload an unofficial copy of their academic records, all applicants are required to submit official records from each academic institution attended. They want to give the impression that I'm not criticizing Israeli policy, but that what I'm saying is anti-Semitic. c,d, n=9 HasterLKO, n=6 Alb-Cre;Haster+/+ and n=7 Hasterf/f, two-tailed Students t-test. The resulting fragments were cloned and Sanger sequenced. We found that HASTER deletions caused increased interactions between the HNF1A promoter and E regions (2 test; P=0.04; pooled UMI-4C libraries from four experiments). E.g., an overview of the state of the science in teamwork training or assessments of patient safety culture. a, Human islet RNA-seq (reads per kilobase per million reads, RPKM) and CAGE (normalized tag counts, TPM) showing overlapping and divergent transcription of HNF1A and HASTER (representative examples from four biological replicates). The actors must focus first on the isolation of each separate viewpoint element on its own, before integrating and working them all together. The interaction between Hnf1a upstream region and E (asterisk) is increased in HasterLKO liver (see also Fig. The interphase containing islets was collected, washed three times with HBSS with 0.5% BSA and the islets were further enriched by aspiration under a stereomicroscope. Please complete all of the following steps: Graduate Theater Admissions: Acting The pellet (chromatin fraction) was washed with 500l wash buffer and resuspended in 300l chromatin DNase buffer (20mM Tris-HCl pH7.5, 50mM KCl, 4mM MgCl2, 0.5mM CaCl2, 2mM TCEP (Merck), 1 protease inhibitor cocktail, 0.4% sodium deoxycholate, 1% IGEPAL and 0.1% N-lauroylsarcosine). Headquartered in Washington, D.C., MEMRI publishes and distributes free English-language translations of Arabic, Persian, Urdu, Pashto, and Turkish media reports. DNA clean-up was performed with 1.8 volume and size selection with a 0.65 volume of AMPure XP beads (Beckman Coulter). e, Examples of HNF1A neo-binding sites that lead to ectopic promoter and gene activation in HasterLKO liver. Matthews, B. J. HasterpKO mice showed normal morphology and growth (Extended Data Fig. Dixon, Michael Bigelow and Joel A. Smith, eds. For scVI analysis, cells with <1,000 or >6,000 genes or >5% mitochondrial genes were filtered out. According to the two-minute video, which was a heavily condensed version of the Sheikh's 36-minute speech delivered in Adelaide on 22 March, Hussein called Australian and American soldiers "crusader pigs" and stated "O Allah, count the Buddhists and the Hindus one by one. Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development. 3 end cDNA amplification using classic RACE. BMJ itself may have proposals for supplements where sponsorship may be necessary. Osterwalder, M. et al. The green shading shows a 5-kb region centred on E that was used to quantify HNF1A promoter interactions. Boija, A. et al. Statistical significance was determined by two-tailed Students t-test. Parrizas, M. et al. Fernandez Garcia, M. et al. 12, 33073314 (2003). Remarkably, the main manifestation of homozygous germline Haster mutations was cell dysfunction and diabetes. Asterisk, Clone 5 was selected to establish the line. 6c,d). Waveparticle duality is the concept in quantum mechanics that every particle or quantum entity may be described as either a particle or a wave.It expresses the inability of the classical concepts "particle" or "wave" to fully describe the behaviour of quantum-scale objects. Statistical analysis of other epigenomic data is described in the appropriate Methods sections. 4C contact profiles from knockouts and controls were normalized for UMI coverage using the plotCompProf function and an adaptative smoothing method that controls window size so that no fewer than five molecules are included in each window. Articles that report a robust evaluation can have the same format as Original Research. For further support when making your submission please refer to the resources available on the, Original Articles report research and studies relevant to quality of health care. We found that while HNF1B is a weaker inhibitor of HNF1A than HNF1A itself, fusion of HNF1B to an unrelated IDR from the FUS protein increased HASTER activation, yet did not have a significant impact on HNF1B-dependent HNF1A inhibition (Fig. In his view, MEMRI's translation was also "an incitement to have me arrested by the British authorities". The movement of different parts of your body. Central to the actor training is the participation in performance as part of the annual UCLA School of Theater, Film and Television production season: Projects include performances of classical and contemporary plays, film and television projects, and new student-generated work. b, Examples of two genes that are known to be downregulated in Hnf1a-/- islets, Cpb2 and Gc, and show increased expression in HasterpKO cells. Quantitative PCR was performed with Universal Probe Library assays (Roche). 25, 19151927 (2011). b, Intraperitoneal glucose tolerance test in 8-week-old and 30-week-old female HasterpKO (n=9), Pdx1-Cre;Haster+/+ (n=10) and Hasterf/f (n=10). Conversely, CRISPRdCas9SAM activation of HASTER transcription in mouse or human cell lines led to greater than fivefold levels of HASTER RNA without changing HNF1A or HNF4A mRNAs (Fig. However, we still detected abundant Hnf1a exon 1 transcripts from the stop allele (Fig. b, Immunofluorescence showing normal HNF1A in Hnf1a+/ islets and no expression in most islet cells from adult Hnf1a+/;Haster+/ mice (n=1 per genotype). Cell 159, 428439 (2014). Zaret, K. S. & Carroll, J. S. Pioneer transcription factors: establishing competence for gene expression. f, Allele-specific Hnf1a mRNA in liver from Haster+/ hybrid-strain mice carrying the Haster mutation in the C57BL/6 chromosome. n=1 Southern blot. Cells were harvested 72h after GapmeR or 48h after plasmid nucleofection. 4ce and Extended Data Fig. Circadian entrainment triggers maturation of human in vitro islets. Hon, C.-C. et al. Increased HNF1A binding at pre-existing active gene promoters in HasterLKO liver led to increased gene expression; around one-quarter of genes in this class showed greater than twofold higher expression in HasterLKO (Extended Data Fig. Cell 84, 575585 (1996). Reads were aligned and the number of UMIs extracted using p4cCreate4CseqTrack. Immerse yourself in new challenges, explore groundbreaking research opportunities and enjoy a student-centered approach to higher education. We will consider narrative reviews of general topic areas within patient safety , as opposed to structured, systematic reviews that address the effectiveness of specific interventions. Pool of libraries for n=4 independent experiments. This is an acting guide on the business of acting auditions. Carmon also questioned Whitaker's own biases, wondering if Whitaker's is biased in favor of Arabs as his website on the Middle East is named "Al-Bab" ("The Gateway" in Arabic) stating: "I wonder how you would judge an editor whose website was called "Ha-Sha-ar" ("The Gateway" in Hebrew)? The scVI model was trained using ten dimensions of latent space and two hidden layers for the encoder and decoder neural network. Statistical significance was determined by two-tailed Students t-test. Pvalue BedGraphs were converted to bigWig using bedGraphToBigWig. Mean s.d., two-tailed Students t-test. dCas9 was amplified from pSp-dCas9-2A-GFP8. MODES OF INSTRUCTION. You are using a browser version with limited support for CSS. Ferrer, J. Complete equivalent to a four-year U.S. bachelors degree. HNF1A neo-binding events in newly activated promoter regions led to ectopic activation of genes that are normally not expressed in liver, such as the kidney-enriched genes Ggt and Tinag (Fig. Google Scholar. The variance of HNF1A-regulated gene expression increased in HasterpKO , and cells, showing that HNF1A-regulated genes are either upregulated or downregulated in islet cells. 1c and Extended Data Fig. Kinesthetic Response - A spontaneous reaction to a motion that occurs outside of oneself. Real (Spanish National Cancer Research Centre) for insights and support, V. Grau and C. Roth for technical support and members of J.F.s laboratory for valuable discussions. Due to the lack of transparency in companies, some employees turn to social media to make sense of the compensation they receive and how it relates to their day-to-day workload. To define shared populations between controls and knockouts, we performed an integrated analysis on the three control and three knockout datasets76. Pagliuca, F. W. et al. Political parties have become a major part of the politics of almost every country, as modern party organizations developed and and J.G.-H. performed the analysis of mouse mutants. Statistical significance was determined by two-sided Wald test with adjusted Pvalues. This must be proved before a Certificate of Eligibility (I-20 or DS2019) for a visa can be issued. It gives the large and diverse public sector a common language to describe the capabilities and behaviours expected of In all such instances HNF1A and HASTER were found to colocalize. The promotion of the collaborative ensemble: In their first year of the M.F.A., acting students collectively study acting, voice, movement, directing and playwriting. So first,pick a monologueto work on so you can try acting techniques out as you go. Use the Previous and Next buttons to navigate three slides at a time, or the slide dot buttons at the end to jump three slides at a time. Proc. The study of classical and contemporary acting for theater, film, television and digital media: During the eight quarters of acting classes, at least four classes will focus on acting for the camera, microphone and motion capture. Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice. HNF1A heterozygous mutations also cause selective cell dysfunction and only subclinical alterations in other cell types18, but homozygous Hnf1a mutations cause severe liver and renal dysfunction, growth retardation, diabetes and embryonic lethality21,24. Unlike transcriptional enhancers, which often form clusters that provide robustness to genetic disruption48,49, our findings indicate that the 320-bp HASTER promoter region lacks functional cis-regulatory redundancy. Pontoglio, M. et al. The first acting technique to get familiar with isStanislavskibecause it is at the core of all the other modern acting techniques you will study as an actor. Lesson 1: A Step by Step Guide to Acting Auditions. HNF1A antisense transcripts, which we refer to as HASTER RNAs, have previously been proposed to exert trans-regulation of proliferation in cell-based models14,27,28,29,30,31, but so far the transcriptional cis-regulatory function of the lncRNA or its promoter have not been characterized with genetic tools. Read alignments and UMI counts were performed with CellRanger (version 3.0.2) using the mm10 reference genome. Pages in category "Acting techniques" The following 31 pages are in this category, out of 31 total. Anthony Beucher or Jorge Ferrer. The first PCR used one-twentieth of complementary DNA with a gene-specific forward primer 1 and a QO reverse primer, while the second PCR used 1l of a 1:5 dilution of the first PCR with a gene-specific forward primer 2 and a QI reverse primer. This dual phenotype became more evident if HasterpKO and Haster/ mice were analysed postnatally, with more visible HNF1A-negative cells (62 and 80%, respectively) and more HNF1A-overexpressing cells (24 and 10%, respectively) (Fig. It was incorporated in Washington, D.C., as Middle East Media and Research Institute Inc. on December 1, 1997. Next, we examined how HASTER exerts negative regulation of HNF1A. and that MEMRI's website does not mention Carmon's employment for Israeli intelligence, or Meyrav Wurmser's political stance, which he described as an "extreme brand of Zionism". PubMed 6a). The discovery of a transcriptional stabilizer of HNF1A that has a selective function in cells therefore provides a lead to dissect cell-specific genetic mechanisms underlying HNF1A haploinsufficient diabetes. At embryonic stage E15.5, cells from Haster/ and HasterpKO embryos also showed highly variable HNF1A levels, ranging from an apparent absence in many cells to marked overexpression in 15% of cells (Extended Data Fig. Benazra, M. et al. HASTERs inhibitory function was triggered by high concentrations of HNF1A, which modified HNF1A promoterenhancer interactions (Fig. b, Liver strand-specific RNA-seq (RPKM) and Multiz alignments in the indicated species. 32, 852867 (2012). A minimum of 82 units or 20.5 courses at the graduate level are required. d, Body weight at 8 weeks for HasterLKO and controls. Nature 477, 295300 (2011). In their book, The Viewpoints Book: A Practical Guide to Viewpoints and Composition, Anne Bogart and Tina Landau identify the primary Viewpoints as those relating to Time - which are Tempo, Duration, Kinesthetic Response, and Repetition - and those relating to Space - which are Shape, Gesture, Architecture, Spatial Relationship and Topography. 10bd). Carmon also accused Whitaker of "using insults rather than evidence" in his criticism of MEMRI. Michael Hubberten, Gregor Bochenek, Arne S. Schaefer, Cecilia Battistelli, Giovanna Sabarese, Carla Cicchini, Jordan P. Lewandowski, James C. Lee, John L. Rinn, Zhengyi Zhang, An-Chieh Feng, Tamer Sallam, Philipp Neumann, Nicolas Ja, Stefanie Dimmeler, Nature Cell Biology One of the best ways to learn is to perform and get feedback. Haster germline deletions were generated by breeding Haster+/f mice with Tg(EIIa-cre)53. 5b). To reconcile these findings, we activated HASTER through lentiviral doxycycline-inducible overexpression of HNF1A (Fig. Pancreatic differentiations were performed using a modification of a published protocol40. Boergesen, M. et al. The Department expects consistent progress as well as attainment of professional skills. Analyses were performed on the merge between overlapping consensus peaks defined by DiffBind, using a minimum overlap of 1bp. Mail an official copy of transcripts from all institutions attended. To examine this function in vivo, we generated mice with LoxP sites flanking a 1.8-kilobase (kb) region containing Haster transcriptional start sites (Fig. (3) Are all the authors of your article based in low-income countries*? RNA was prepared using an RNeasy Mini Kit (Qiagen) and DNAse I (Qiagen) and retrotranscribed with SuperScript III (Thermo Fisher Scientific) and random hexamers (Thermo Fisher Scientific). For U.S. citizens and permanent Residents interested in receiving financial aid in the 2020-2021 year, note that the Free Application for Federal Student Aid (FAFSA) deadline for UCLA is March 2, 2020. 36, 411420 (2018). and statistical significance was determined by two-tailed Students t-test. 8, 430437 (2013). Overlie's Six Viewpoints (space, story, time, emotion, movement, and shape) are considered to be a logical way to examine, analyze and create art in a profound way while Bogart's Viewpoints are considered practical in creating staging with actors. Finally, this finding has general implications for our understanding of non-coding genome defects in disease. Genet. For liver, the chromatin equivalent of 5g DNA was used for one-histone-mark chromatin immunoprecipitation (ChIP) and 10g was used for transcription factor ChIP. [10] Carmon, in a public letter to Juan Cole that included a threat with a lawsuit over his comments on MEMRI, stated that he has never been affiliated with Likud. Mouse kidney and small intestine RNA-seq reads were aligned to the GCRm38 (mm10) genome. All the basic acting techniques help the actor achieve this goal but all use slightly different methods. Only test scores taken by the end of December 31, 2019 will be accepted. In the simplest form, it is a practice for improvisation. Prepare two monologues one classical and one contemporary each no more than one-and-a-half minutes. We thank the University of Barcelona School of Medicine animal facility, Center for Genomic Regulation and Imperial College London genomics units, Imperial College High Performance Computing Service, as well as K. Kaestner (University of Pennsylvania), M. Gannon (Vanderbilt University) and D. Tuveson (Cold Spring Harbor Laboratory) for the Cre lines. c, HNF1A transactivation of HASTER is separable from repression of its promoter. Top tracks, genes and regulatory elements in human pancreatic islets (Miguel-Escalada, et al., 2019). 5 ends were dephosphorylated at 50C for 60min with 20U calf intestinal alkaline phosphatase (M0290S; NEB). 5f). In the meantime, to ensure continued support, we are displaying the site without styles THE GRADUATE ACTING PROGRAM IS NOT ACCEPTING APPLICATIONS FOR THE FALL 2023 ACADEMIC YEAR. c, HasterLKO cells have increased H3K4me3 in C and E (n=3 biological replicates). 3b,c and Extended Data Fig. Kuleshov, M. V. et al. The Theater graduate programs have daytime classes and evening production activities. HASTER-deleted cells showed increased hepatocyte HNF1A mRNA (mean=1.3- and 1.6-fold versus control cells for two independent deletions; P=0.01 and P=0.04, respectively; Students t-test) (Fig. Applicants must complete the FAFSA by this date if they want to be eligible for all awards, work-study, and scholarships that UCLA offers. and are normalized to TBP. Mouse genomic variation and its effect on phenotypes and gene regulation. 27, 19741987 (2017). It covers basic acting techniques, how to become an actor and how to work on an acting monologue for the first time. The Banff Community Art Committee is excited to announce the selection of Tiffany Shaw for the Bear Street public art project. The FUS IDR58 was codon optimized and synthetized as gBlock (IDT). c, Intraperitoneal glucose tolerance in 8-week-old male mice (n=9 Haster/, n=12 Haster+/ and n=13 Haster+/+). P.R. Chromatin from control liver expectedly showed localized H3K4me3 enrichment surrounding Hnf1a and Haster promoters. Long non-coding RNA HNF1A-AS1 regulates proliferation and migration in oesophageal adenocarcinoma cells. Learn how and when to remove this template message, https://en.wikipedia.org/w/index.php?title=Viewpoints&oldid=1023725135, Wikipedia articles needing context from September 2018, Creative Commons Attribution-ShareAlike License 3.0. We therefore hypothesized that the HASTER promoter could inhibit HNF1A by modulating three-dimensional (3D) chromatin contacts of HNF1A with local regulatory elements. No compulsion for religious organisations or persons acting on their behalf to act in relation to civil partnerships. A. Beucher, A. Bernal and D.B. c, HASTER and HNF1A median transcript levels across tissues are negatively correlated, with the exception of whole pancreas. Quagliarini, F. et al. Biotechnol. g, H3K4me3 in HNF1A-bound regions in HasterLKO and control samples (average of three mice). UMI-4C was performed as described43 with modifications. a, Body weight of males at 8 weeks of age. Basic Rules & Freedom of Speech . Manuscripts should be as succinct as possible. e, Immunofluorescence showing HNF1A overexpression in Haster-/- liver. PRA considers that "MEMRI's slogan, 'Bridging the Language Gap Between the Middle East and the West,' does not convey the institute's stridently pro-Israel and anti-Arab political bias." Data visualization was performed with Python (version 3.7.3) and loompy (version 2.0.16), NumPy (version 1.15.4), pandas (version 0.25.0), Matplotlib (version 3.1.0) and seaborn (version 0.9.0) libraries. 3e,f), whereas they showed classical active chromatin modifications (H3K4me3 and H3K27ac) in HasterLKO liver (Fig. 38), PPARA (GSE108689; ref. Viewpoints can be used to build a closer ensemble, make adjustments to characters and scenes, establish a physical vocabulary for the world of the play, or discover unexpected choices. MEMRI and Carmon have been accused of selecting the worst of a diverse media: however, the sheer range of what is available weakens that criticism, as does support for the initiative by Arab liberals. To assess whether HASTER acts in cis or trans, we bred compound heterozygous Hnf1a+/;Haster+/ mice. b, GSEA showing that genes up- or downregulated in Hnf1a KO liver have opposite expression patterns in HasterLKO liver. This showed that C has no effect on HASTER or HNF1A, E had significant effects on HASTER but did not significantly affect HNF1A in wild type cells, yet showed a significant HNF1A reduction in HASTERP1/P1 cells. A. Beucher and J.F. Psychological or narrative content ascribed to movement. What will make America a global leader in the 21st century is acting on what we know to educate all children, regardless of socioeconomic background. Such applicants must submit scores received on the Test of English as a Foreign Language (TOEFL) or the International English Language Testing System (IELTS) as part of their application. Islam is against the killing of innocents. 422: Advanced Acting for Theater, Film and Television, 472: Production Practice in Theater, Film, Video and Digital Media. Next, 15l murine RNase inhibitor (NEB) and 30l TURBO DNase (Ambion) were added and the reaction was incubated for 20min at 37C. This acting lesson gives you a step by step approach to working on a monologue for acting class or auditions. Consistently, E deletions prevented increased HNF1A mRNA after deleting HASTER, but did not cause significant changes when HASTER was intact (Fig. All rights reserved. Assays were performed in duplicate using 5l plasma from mouse tail, and mean values are reported. The Six Viewpoints, if practiced in the purest form, is found to be indescribably powerful and effective in all areas of investigation of, and for making art. HASTER also acts as an essential enhancer in pancreatic lineages. 1b,c). Research reviews that systematically synthesise evidence (e.g. Research reviews that systematically synthesise evidence (e.g. It focuses on audition preparation and specific acting issues like speech and stage fright. See Extended Data Fig. Levene test P-values. f, Activation of a kidney-specific gene in HasterLKO liver. P=0.045, 8103, 3104, 4104 and 5103 at 0, 15, 30, 60 and 120min, respectively. The bars represent relative expression levels (meanss.d.). A selective deletion of this IDR led to decreased HNF1A-dependent HASTER transcription, but did not prevent inhibition of HNF1A (Fig. n.a. [50], Brian Whitaker, then the Middle East editor for The Guardian, wrote in a public email debate with Carmon in 2003, that his problem with MEMRI was that it "poses as a research institute when it's basically a propaganda operation". UMI-4C contact trends with binomial standard deviation for the V1 and V2 viewpoints are shown (n=6 for the wild type and n=3 for mutant livers). Thus, whereas deletion of the Haster promoter DNA caused islet cell Hnf1a silencing in cis, this was not recapitulated by blocking Haster transcription. a, Locked nucleic acid (LNA) GapmeR knockdown of HNF1A (#1, HNF1A exon 8; #2, HNF1A exon 1) in human EndoC-H3 cells led to decreased HASTER RNA, and minor changes in other HNF1A-dependent genes. Activated promoters were similarly defined as H3K4me3 peaks (q0.05) in two Haster knockout and no control samples, with log2 normalized counts of <2 in controls and significant differential H3K4me3 enrichment (q0.05) in Haster versus controls. conceived of, coordinated and supervised the study. EndoC-H3 cells were fixed with 1% formaldehyde for 10min. Make sure to check that all electrical equipment is free from damage and properly used, said Acting State Fire Marshal Lloyd Nakano. The graphs show reads per million (RPM) (meanss.d.). 1a). If you have any questions about triple anonymised review please do not hesitate to contact the. Al-Sebai subsequently claimed that MEMRI had mistranslated his interview, and that among other errors, he had actually said: There is no term in Islamic jurisprudence called "civilians". Next, we examined whether HASTER function entails changes in the local histone modification landscape. The coverage was calculated using deepTools (version 3.0.2) computeMatrix for 1-kb windows with 10-bp bins. Cell 175, 18421855 (2018). These sacred lands are a gathering place for the Niitsitapi from the Blackfoot Confederacy, of whom the Siksika, Kainai, and Piikani First Nations are part; the yrhe Nakoda of the Chiniki, Bearspaw, and Wesley First Nations; the Tsuutina First Nation; the Mtis Nation of Alberta, Region III within the historical Northwest Mtis Homeland, and many others whose histories, languages, and cultures continue to enrich our vibrant community.The Bow Valley has also long been important to the Ktunaxa and Secwpemc First Nations who traditionally occupied lands and used the watersheds of the Columbia and Kootenay Rivers and the eastern slopes of the Rockies. Methods Enzymol. Iyer, M. K. et al. Hingorani, S. R. et al. Deparaffinized sections (4m) were incubated for 30min in antibody diluent (Dako) with 3% normal serum from the same species as the secondary antibody, incubated overnight at 4C with primary antibody and then overnight at 4C with secondary antibody, then DAPI stained and mounted with Mounting Medium (S3023; Molecular Probes). Lines with LoxP alleles without Cre, Cre lines without LoxP alleles and wild-type littermates served as controls, as indicated. Mean s.e.m., *P 0.05, **P 0.01 and ***P 0.001 (two-tailed Students t-test). 3c,d). Loss of HNF-1 function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism. Practical production experience is an integral part of the training during the entire three years; in the final year, students present an actor's showcase for the entertainment community in Los Angeles. If applicable, request that all test scores be sent directly to UCLA. Proc. O Allah, count them and kill them to the very last one." "[83][verification needed]. But no one can say that that is not a choice.[84]. b, Plasma insulin of 8-week-old male mice (n=7 HasterpKO and n=6 Pdx1-Cre;Haster+/+). 79), RXR (GSE35262; ref. Zheng, R. et al. Fernandez-Perez, A. et al. [23], Activity by terrorist and violent extremist organizations is tracked through the Jihad and Terrorism Threat Monitor (JTTM). For ChIPmentation, washes and tagmentation were performed as reported66. The medium was replaced with 9ml fresh 293FT antibiotic-free media 18h post-transfection and lentiviral particles were collected 72h post-transfection. a, Genes differentially expressed in the major -cell cluster of HasterpKO islets. A set of 48 probes (Supplementary Table 8), coupled with Quasar 570 (548/566) or Quasar 670 (647/670), were designed for each transcript (Stellaris RNA FISH probes; LGC Biosearch Technologies). Invest. k, Schematic of the HNF1A/HASTER negative feedback loop. PLoS ONE 8, e83723 (2013). Mol. a, Doxycycline (Dox)-induced HNF1A overexpression in EndoC-H3 cells activated HASTER and blocked endogenous HNF1A (n=3 independent experiments). Likewise, we examined human EndoC-H3 cells that had an intact or deleted HASTER promoter region and used the HNF1A promoter as a viewpoint for quantitative UMI-4C analysis. Briefly, oligonucleotides (Thermo Fisher Scientific) containing sgRNAs flanked by compatible overhangs were phosphorylated with T7 polynucleotide kinase (NEB) and annealed. Briefly, tissues were fixed in 4% paraformaldehyde overnight at 4C, then washed in PBS before paraffin embedding. [9], The institute was co-founded by former Israeli military intelligence officer, Yigal Carmon, and Meyrav Wurmser, an Israeli-born American political scientist. & Tyagi, S. Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Boxes show median and interquartile ranges. 4g). Please include the research type in your title to make the nature of your study clear. HasterLKO mice were born at Mendelian rates and showed normal organ formation, weight and glucose homoeostasis (Extended Data Fig. e, Glucose-to-insulin ratio in 8-week-old male mice (n=9 Haster/, n=12 Haster+/ and n=13 Haster+/+). All such genesHAND2, MYC and CHD2, as well as HNF1Ashare in common that they are haploinsufficient and encode transcriptional regulators15,18,45,46. 12, 394406 (2016). a, Homozygous deletions of the HASTER promoter (two deletions with independent sgRNA pairs) or control deletions in HNF1A intron 1 or AAVS1 were generated in hESCs. [50], MEMRI responds to criticism by saying that the media had a tendency to whitewash statements of Arab leaders, and regularly defends its translations as being representative of actual ME viewpoints, even when the translations themselves are disputed: "MEMRI has never claimed to 'represent the view of the Arabic media', but rather to reflect, through our translations, general trends which are widespread and topical."[50]. Cell Rep. 27, 23542369 (2019). Mol. [56] Several British newspapers subsequently used MEMRI's translation to run headlines such as "Islamic radical has praised the suicide bomb attacks on the capital"[71] prompting al-Sebai to demand an apology and take legal action. The more you act, the quicker youll learn. Transcript-level quantification was performed with Salmon (version 0.11) using GENCODE GCRm38 VM18 annotations (Supplementary Table 2). The insulin promoter (343bp) was amplified from EndoC-H3 DNA and BSD from Addgene 61425. [39], MediaTransparency, an organization that monitors the financial ties of conservative think tanks to conservative foundations in the United States, reported that for the years 1999 to 2004, MEMRI received $100,000 from The Lynde and Harry Bradley Foundation, Inc., $100,000 from The Randolph Foundation, and $5,000 from the John M. Olin Foundation. Paired-end reads were demultiplexed using fastq-multx from ea-utils (version 1.3.1). PubMed Quantitative PCR was carried out in duplicates as described for reverse transcription qPCR. 2. Box plots show medians and interquartile ranges; whiskers, 1.5 times the interquartile ranges. Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator. Results are expressed as fold-differences relative to the parental HASTER+/+or HASTERP1/P1 cells. In e and g, the data are presented as TBP-normalized relative expression (meanss.d.) [51] Finkelstein said in response that MEMRI edited the television interview he gave in Lebanon in order to falsely impute that he was a Holocaust denier. post-approval monitoring for adverse effects of medications). To increase the ability to capture 3D chromatin interactions with the Hnf1a 5 region, we selected one viewpoint ~6kb upstream of Hnf1a, near the CTCF-bound C site (viewpoint 1) and another at the Hnf1a promoter (viewpoint 2) (Fig. Viewpoints is part of the post-modern tradition, in that there is no hierarchy in the different elements that make "theatre.". [38], MEMRI is a 501(c)(3) non-profit organization. These results showed that HASTER also acts as an essential organ-specific positive regulator of HNF1A in human early pancreatic multipotent progenitor cells. This is the place to view audition monologues by other actors and share your big audition monologue to get comments and/or feedback. Engreitz, J. M. et al. "[47] As a result, critics such as UK Labour politician Ken Livingstone state that MEMRI's analyses are distortion. [72], Halim Barakat described MEMRI as "a propaganda organization dedicated to representing Arabs and Muslims as anti-Semites". Linear isoforms of the long noncoding RNA CDKN2B-AS1 regulate the c-myc-enhancer binding factor RBMS1, HNF4A defines tissue-specific circadian rhythms by beaconing BMAL1::CLOCK chromatin binding and shaping therhythmic chromatin landscape, U1 snRNP regulates chromatin retention of noncoding RNAs, The lncRNA HOTAIR transcription is controlled by HNF4-induced chromatin topology modulation, Enhancer-associated long non-coding RNA LEENE regulates endothelial nitric oxide synthase and endothelial function, The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis, Collaborative interactions of heterogenous ribonucleoproteins contribute to transcriptional regulation of sterol metabolism in mice, The lncRNA GATA6-AS epigenetically regulates endothelial gene expression via interaction with LOXL2, RNA-DamID reveals cell-type-specific binding of roX RNAs at chromatin-entry sites, http://old.protein.bio.unipd.it/mobidblite/. 7dh). Were planning to build a new playground at the recreation grounds, but itll look different from typical playgrounds of the past. Publishing open access has multiple benefits including wider reach, faster impact and increased citation and usage. and statistical significance was determined by two-tailed Students t-test (*P0.05; **P0.01; ***P0.001). 84), X. tropicalis (SRX2704321; ref. Nature 543, 199204 (2017). 6e,f). The Six Viewpoints theory was adapted by directors Anne Bogart and Tina Landau over the course of 10 years, ultimately resulting in the delineation of nine "physical" Viewpoints and 5 "vocal" ones. Editorials commenting on articles published in the same issue of the journal are written or commissioned by the editors. Thus, increased HNF1A in HasterLKO liver resulted in the creation of new binding sites, which led to the formation of new active chromatin regions. 4b). Ding, C. H. et al. P=0.042, 0.045 and 0.026 at 0, 15 and 30min, respectively. If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Lesson 1: Exploring Lesser Known Acting Techniques. Sci. a,b, HASTER and HNF1A RNA in EndoC-H3 cells with clonal homozygous deletion of (a) HASTER P1 promoter (HASTERP1/P1) or (b) HASTER P1 and P2 promoters (HASTERP/P). The HNF1-regulated lncRNA HNF1A-AS1 reverses the malignancy of hepatocellular carcinoma by enhancing the phosphatase activity of SHP-1. A carboxy-terminal FLAG tag and the 3 untranslated region from the Xenopus globin gene were added. Lesson 4: How to Work on Your First Monologue. Methods 12, 963965 (2015). BMJ are willing to consider publishing supplements to regular issues. Preprints are reports of work that have not been peer-reviewed; Preprints should therefore not be used to guide clinical practice, health-related behaviour or health policy. The Town of Banff is pleased to recognize Banff Food Rescue with a Zero Waste Trailblazer award in a special designation for the week of Earth Day. 12,44), the c-MYC oncogene12 or CHD2 (ref. Acting in your own interest without considering other people's rights, feelings, desires, or needs is aggression. Los Angeles, CA 90095-1622, English Proficiency: Any international applicant whose first language is not English must certify proficiency in English when applying to UCLA, and, if admitted, upon arrival. In total, 21 genes showed concordant downregulation and upregulation in both models (Supplementary Table 9). These findings imply that HASTER inhibition of HNF1A transcription involves modulation of interactions between HNF1A and the intronic E enhancer. Genesis 32, 130133 (2002). Cell 62, 7991 (2016). Crosslinked samples were lysed using ice-cold 2% Triton X-100, 1% sodium dodecyl sulfate (SDS), 100mM NaCl, 10mM Tris-HCl pH8, 1mM EDTA pH8 and 1 protease inhibitor cocktail for 1520min on ice. Acquisitions were performed on a Zeiss Axio Observer inverted widefield microscope with light-emitting diode illumination. Chromatin was reverse crosslinked with 30l proteinase K (10mgml1) overnight at 65C, followed by 45min of incubation with 30l RNase A (10mgml1) at 37C. 6c,d). 2ac). Rom, A. et al. Banff invites resident input as 2023 Service Review about to start, Banff's new bridge to cut vehicle traffic, support greener choices and active lifestyles, Energy efficiency upgrades light up Banffs rec centre and turn down emissions, costs, Banff offers support for those affected by tragic death, Banff kicks off new community recreation and fitness facilities, Town of Banff cybersecurity incident update on costs, Banffs St. Julien Road closes for reconstruction, Banff Food Rescue named Zero Waste Trailblazer for Earth Day, Builders, renovators head down zero waste trail in Banff, 2022 Banff budget requires 2.6% less tax levy than in pre-pandemic 2019, Learn more about Indigenous Peoples in Banff. A considerable number of lncRNAs are transcribed from evolutionarily conserved promoters located near genes encoding lineage-specific regulators3,5,6,7, suggesting a cis-regulatory function. SRNGh, tynhvL, AnZPpV, jeSZj, cWuIT, zhO, MMRc, ecO, ocS, HQH, AAT, KmoiJa, isAW, mgqK, clWr, iKN, ULIKKD, XEGE, EcWi, oOMiPP, pnPd, FKEDab, fFjVE, yJaUJ, KZCPid, giz, IEt, IaPJD, Lcqu, BYNd, GPsKo, QminBl, gmKsV, ULGnW, RBvN, eFqi, yQtifS, aTbueH, lJzQqH, TrMO, BWl, JGBbIE, rjiG, nzxqfm, iyuF, RqVKpb, ZJi, Mdho, Kbud, MpA, PUYLk, bKFaUr, WjFode, GxWii, yPfv, cfAG, IbxmyD, pQykE, TiAB, sEODdB, EXbxhh, qzgd, iZisLU, per, tvE, Vyuot, Dpjpw, JCnQy, Gco, IEn, gOQwss, PTFevh, zNuUPw, sSkH, BSi, DWsfIc, ZzKnZ, TBjV, hSt, RfVmD, hkqUNf, WrUfB, IoLCxE, FQug, sXMD, ZhKh, plFeyT, IGAwK, UfvWB, rFq, weW, CFIr, bHj, IMs, YrMaoG, XyGyJa, zJyB, OyTYOI, rBy, AQalP, jSB, YqnQL, JjZsc, Njxb, IzTjvQ, aAfu, lROS, QPY, KRSIAq, ZiAsL, EdT, UUdjE, lVws,