Design Parameters to Control Synthetic Gene Expression in Escherichia coli

BACKGROUND:Production of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles. PRINCIPAL FINDINGS:To identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well. CONCLUSION:The systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system

Surgical Techniques to Optimize Early Urinary Continence Recovery Post Robot Assisted Radical Prostatectomy for Prostate Cancer.

PURPOSE OF REVIEW: A variety of different surgical techniques are thought to impact on urinary continence (UC) recovery in patients undergoing robot assisted radical prostatectomy (RARP) for prostate cancer. Herein, we review current evidence and propose a composite evidence-based technique to optimize UC recovery after RARP. RECENT FINDINGS: A literature search on studies reporting on surgical techniques to improve early continence recovery post robotic prostatectomy was conducted on PubMed and EMBASE. The available data from studies ranging from randomized control trials to retrospective cohort studies suggest that minimizing damage to the internal and external urinary sphincters and their neural supply, maximal sparing of urethral length, creating a secure vesicourethral anastomosis, and providing anterior and posterior myo- fascio-ligamentous support to the anastomosis can improve early UC recovery post RARP. A composite evidence-based surgical technique incorporating the above principles could optimize early UC recovery post RARP. Evidence from randomized studies is required to prove benefit

Peri-operative, functional and early oncologic outcomes of salvage robotic-assisted radical prostatectomy after high-intensity focused ultrasound partial ablation

BACKGROUND: Partial ablation of the prostate using high-intensity focussed ultrasound (HIFU-PA) is a treatment option for localised prostate cancer. When local recurrence occurs, salvage robot-assisted radical prostatectomy is a treatment option for selected patients, but there is a paucity of data on the peri-operative safety, functional and oncologic outcomes of sRARP.. The objective of this study was therefore to describe peri-operative safety, functional and early oncologic outcomes following salvage robot-assisted radical prostatectomy (sRARP) for local recurrence after HIFU-PA. METHODS: Retrospective analysis of a prospective database of 53 consecutive men who underwent sRARP after HIFU-PA from 2012 to 2018. Continence and erectile-function were reported pre-HIFU, pre-sRARP, 3-months post-sRARP and 12-months post-sRARP. Complications, PSMs and need for subsequent ADT/radiotherapy were assessed. RESULTS: 45 men were suitable for inclusion and had sufficient data for analyses. Median duration from HIFU to sRARP was 30.0 months and median follow-up post-sRARP was 17.7 months. Median age, PSA and ISUP group were 63.0 yrs., 7.2 ng/mL and 2; 88.9% were cT2. Median operative-console time, blood loss and hospital stay were 140 min, 200 ml and 1 day respectively. Clavien-Dindo grade 1, 2 and 3 complications 90d) complications occurred in 13.2%. At sRARP pathology, ISUP 3-5 occurred in 51.1%, pT3a/b in 64.5%, and PSMs in 44.4% (37.5% for pT2, 48.3% for pT3). Of men with > 3-months follow-up after sRARP, 26.3% underwent adjuvant radiotherapy/ADT for residual disease or adverse pathologic features; 5.3% experienced BCR requiring salvage ADT/radiotherapy. Freedom from ADT/radiotherapy was 66.7% at 12-months. Pad-free rates were 100% pre-HIFU, 95.3% post-HIFU, 29.4% 3-months post-sRARP, and 65.5% 12-months post-sRARP. Median IIEF-5 scores pre-HIFU, post-HIFU, 3- and 12-months post-sRARP were 23.5, 16, 5 and 5, respectively. Potency rates were 81.8, 65.5, 0 and 0%, respectively. Bilateral/unilateral nerve sparing were feasible in 7%/22%. CONCLUSION: Salvage RARP was safe with acceptable but sub-optimal continence and poor sexual-function and poor oncologic outcomes. One in three men required additional treatment within 12-months. This information may aid men and urologists with treatment selection and counselling regarding primary HIFU-PA vs primary RARP and when considering salvage RARP

Presentations at the Flosolver Workshop :27-28 December 1988

The -transparencies used in the two -day 'workshop held at NAL on 27-28 December 1988 have been on Flosolver reproduced

Sub-100 nm CMOS circuit performance with high-K gate dielectrics

In this paper we look at the effect of fringing fields on the circuit performance by use of high permittivity (K) gate dielectrics in 70 nm CMOS technologies, from Monte-Carlo and mixed-mode simulations. Our results clearly show a decrease in the external fringing capacitance and an increase in the internal fringing capacitance, when the conventional SiO2 is replaced by high-K gate dielectrics. It also indicates an optimum K value for a given technology generation in terms of circuit and device short-channel performance. (C) 2001 Elsevier Science Ltd. All rights reserved

Preliminary report on the parallelisation of the panel code on Flosolver

A Panel Code from SOFFTS has been parallelised and the advantage of parallel computing in terms of computing speed has been clearly demonstrated13;

Failure analysis of centre glass panel of cockpit of a helicopter

Fractographic study showed that the glass panels have failed by fatigue wherein the fatigue cracks have initiated at the bolt cutout surfaces. Comparative study carried out showed that although both imported and indigenous glass panels are made of same polymer (PMMA), the imported one has a wider molecular weight distribution (MWD) than that of the indigenous one. Fatigue tests carried out on the samples taken from these glass panels showed that the former has superior fatigue crack resistance than that in the latter. It is believed that because of wide MWD, the imported glass panel has better fatigue resistance than that of the indigenous one. A detailed analysis of the study is presented in this report