CONSERVED FUNCTIONAL DOMAINS OF THE RNA-POLYMERASE-III GENERAL TRANSCRIPTION FACTOR BRF

In Saccharomyces cerevisiae, two components of the RNA polymerase III (Pol III) general transcription factor TFIIIB are the TATA-binding protein (TBP) and the B-related factor (BRF), so called because its amino-terminal half is homologous to the Pol II transcription factor IIB (TFIIB). We have cloned BRF genes from the yeasts Kluyveromyces lactis and Candida albicans, Despite the large evolutionary distance between these species and S. cerevisiae, the BRF proteins are conserved highly. Although the homology is most pronounced in the amino-terminal half, conserved regions also exist in the carboxy-terminal half that is unique to BRF. By assaying for interactions between BRF and other Pol III transcription factors, we show that it is able to bind to the 135-kD subunit of TFIIIC and also to TBP. Surprisingly, in addition to binding the TFIIB-homologous amino-terminal portion of BRF, TBP also interacts strongly with the carboxy-terminal half. Deleting two conserved regions in the BRF carboxy-terminal region abrogates this interaction. furthermore, TBP mutations that selectively inhibit Pol III transcription in vivo impair interactions between TBP and the BRF carboxy-terminal domain. Finally, we demonstrate that BRF but not TFIIB binds the Pol III subunit C34 and we define a region of C34 necessary for this interaction. These observations provide insights into the roles performed by BRF in Pol III transcription complex assembly

Repair of Aberrant Splicing in Growth Hormone Receptor by Antisense Oligonucleotides Targeting the Splice Sites of a Pseudoexon

Context: The GH receptor (GHR) pseudoexon 6 Psi defect is a frequent cause of GH insensitivity (GHI) resulting from a non-functioning GH receptor (GHR). It results in a broad range of phenotypes and may also be present in patients diagnosed as idiopathic short stature.Objective: Our objective was to correct aberrant GHR splicing and inclusion of 6 Psi using exon-skipping antisense oligonucleotides (ASOs).Design and Setting: Three ASOs binding the 5' (ASO-5), 3' (ASO-3), and branch site (ASO-Br) of 6 Psi were tested in an in vitro splicing assay and a cell transfection system. The wild-type (wt) and mutant (mt) DNA minigenes (wt- and mtL1-GHR6 Psi-L2, respectively) were created by inserting the GHR 6 Psi in a well-characterized splice reporter (Adml-par). For the in vitro splicing assay, the wt- and mtL1-GHR6 Psi-L2 were transcribed into pre-mRNA in the presence of [alpha P-32]GTP and incubated with ASOs in HeLa nuclear extracts. For the cell transfection studies, wt-and mtL1-GHR6 Psi-L2 cloned into pcDNA 3.1 were transfected with ASOs into HEK293 cells. After 48 h, RNA was extracted and radiolabeled RT-PCR products quantified.Results: ASO-3 induced an almost complete pseudoexon skipping in vitro and in HEK293 cells. This effect was dose dependent and maximal at 125-250 nM. ASO-5 produced modest pseudoexon skipping, whereas ASO-Br had no effect. Targeting of two splice elements simultaneously was less effective than targeting one. ASO-Br was tested on the wtL1-GHR6 Psi-L2 and did not act as an enhancer of 6 Psi inclusion.Conclusions: The exon-skipping ASO approach was effective in correcting aberrant GHR splicing and may be a promising therapeutic tool. (J Clin Endocrinol Metab 95: 3542-3546, 2010

Vapor condensation on a turbulent liquid interface

An experimental investigation which seeks the fundamental relationship between the interfacial condensation rate and the parameters which control it when the liquid side is turbulent is discussed. The scaling laws for free-surface condensation are discussed for this case. It is argued that the condensation of cryogenic liquids can, in principle, be simulated in experiments using steam and water. Data are presented for the condensation rate in terms of the dimensionless scaling parameters which involve the fluid properties and the liquid-side turbulence velocity and length scales

An Alternative Q Chart Incorporating A Robust Estimator Of Scale

In overcoming the shortcomings of the classical control charts in a short runs production, Quesenberry (1991 & 1995a – d) proposed Q charts for attributes and variables data. An approach to enhance the performance of a variable Q chart based on individual measurements using a robust estimator of scale is proposed. Monte carlo simulations are conducted to show that the proposed robust Q chart is superior to the present Q chart

A Modified \u3cem\u3eX̄\u3c/em\u3e Control Chart for Samples Drawn from Finite Populations

The X̄ chart works well under the assumption of random sampling from infinite populations. However, many process monitoring scenarios may consist of random sampling from finite populations. A modified X̄ chart is proposed in this article to solve the problems encountered by the standard X̄ chart when samples are drawn from finite populations

Splicing therapeutics in SMN2 and APOB

Splicing therapeutics are defined as the deliberate modification of RNA splicing to achieve therapeutic goals. Various techniques for splicing therapeutics have been described, and most of these involve the use of antisense oligonucleotide-based compounds that target key elements in the pre-mRNA to control splicing in the nucleus. In this review, recent developments in splicing therapeutics for the treatment of two specific diseases are described: correcting the alternative splicing of survival of motor neuron (SMN)2 pre-mRNA to compensate for the defective SMN1 gene in spinal muscular atrophy, and re-engineering the splicing of apolipoprotein B pre-mRNA to lower circulating cholesterol levels

Validation of the English and Chinese versions of the Quick-FLIC quality of life questionnaire.

A useful measure of quality of life should be easy and quick to complete. Recently, we reported the development and validation of a shortened Chinese version of the Functional Living Index-Cancer (FLIC), which we called the Quick-FLIC. In the present study of 327 English-speaking and 221 Chinese-speaking cancer patients, we validated the English version of the Quick-FLIC and further assessed the Chinese version. The 11 Quick-FLIC items were administered alongside the 11 remaining items of the full FLIC, but there appeared to be little context effect. Validity of the English version of the Quick-FLIC was attested by its strong correlation with two other measures of quality of life, and its ability to detect differences between patients with different performance status and treatment status (each P<0.001). Its internal consistency (alpha=0.86) and test-retest reliability (intraclass correlation=0.76) were also satisfactory. The measure was responsive to changes in performance status (P<0.001). The Chinese version showed similar characteristics. The Quick-FLIC behaved in ways that are highly comparable with the FLIC, even though the Quick-FLIC comprised only 11 items whereas the FLIC comprised 22. Further research is required to see whether the use of shorter instruments can improve data quality and response rates, but the fact that shorter instruments place less burden on the patients is itself inherently important

DNA methylation of ESR-1 and N-33 in colorectal mucosa of patients with Ulcerative Colitis (UC)

Introduction: Epigenetic marking such as DNA methylation influence gene transcription and chromosomal stability and may also be affected by environmental exposures. Few studies exist on alteration in DNA methylation profiles (genomic and gene specific methylation) in patients with Ulcerative Colitis (UC) and none assessing its relationship with lifestyle exposures. Aims & Methods: To assess genomic methylation and promoter methylation of the ESR-1 (oestrogen receptor - 1) and N-33 (tumour suppressor candidate-3) genes in the macroscopically normal mucosa of UC patients as well as to investigate effects of anthropometric and lifestyle exposures on DNA methylation. Sixty eight subjects were recruited (24 UC and 44 age and sex matched controls). Colorectal mucosal biopsies were obtained and DNA was extracted. Genomic DNA methylation was quantified using the tritium-labelled cytosine extension assay (3[H] dCTP) whilst gene specific methylation was quantified using the COBRA method. Results: The methylation level of both ESR-1 and N-33 genes were significantly higher in UC subjects compared with controls (7.9% vs 5.9%; p = 0.015 and 66% vs 9.3%; p < 0.001 respectively). There was no detectable difference in global DNA methylation between patients with UC and age and sex matched controls. No associations between indices of DNA methylation and anthropometric measures or smoking patterns were detected. Conclusions: For the first time, we have shown increased methylation in the promoter regions of the putative tumour suppressor gene N-33 in macroscopically normal mucosa of patients with UC. In addition, we have confirmed that methylation of ESR-1 promoter is higher in UC patients compared with age and sex matched controls. These findings suggests that, inactivation through methylation of the putative tumour suppressor genes N-33 and ESR-1, may not be associated with colorectal carcinogenesis in UC