33 research outputs found
Recommended from our members
GAP WORK project report: training for youth practitioners on tackling gender-related violence
This project sought to challenge gender-related violence against (and by) children and young people by developing training for practitioners who have everyday contact with general populations of children and young people (‘youth practitioners’). Through improved knowledge and understanding practitioners can better identify and challenge sexist, sexualising, homophobic or controlling language and behaviour, and know when and how to refer children and young people to the most appropriate support services. This summary outlines the Project and our initial findings about the success of the four training programmes developed and piloted.Co-funded by the DAPHNE III programme of the EU
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
Expression Screening of Fusion Partners from an E. coli Genome for Soluble Expression of Recombinant Proteins in a Cell-Free Protein Synthesis System
While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes.National Research Foundation of KoreaKorea (South). Ministry of Education, Science and Technology (MEST) (grant 2011K000841)Korea (South). Ministry of Education, Science and Technology (MEST) (grant 2011-0027901
Effect of combined treatment with alendronate and calcitriol on femoral neck strength in osteopenic rats
<p>Abstract</p> <p>Background</p> <p>Hip fracture is associated with pronounced morbidity and excess mortality in elderly women with postmenopausal osteoporosis. Many drugs have been developed to treat osteoporosis and to reduce the risk of osteoporotic fractures. We investigated the effects of combined alendronate and vitamin D<sub>3 </sub>treatment on bone mass and fracture load at the femoral neck in ovariectomized (OVX) rats, and evaluated the relationship between bone mass parameters and femoral neck strength.</p> <p>Methods</p> <p>Thirty 12-week-old female rats underwent either a sham-operation (n = 6) or OVX (n = 24). Twenty weeks later, OVX rats were further divided into four groups and received daily doses of either saline alone, 0.1 mg/kg alendronate, 0.1 μg/kg calcitriol, or a combination of both two drugs by continuous infusion via Alzet mini-osmotic pumps. The sham-control group received saline alone. After 12 weeks of treatment, femoral necks were examined using peripheral quantitative computed tomography (pQCT) densitometry and mechanical testing.</p> <p>Results</p> <p>Saline-treated OVX rats showed significant decreases in total bone mineral content (BMC) (by 28.1%), total bone mineral density (BMD) (by 9.5%), cortical BMC (by 26.3%), cancellous BMC (by 66.3%), cancellous BMD (by 29.0%) and total cross-sectional bone area (by 30.4%) compared with the sham-control group. The combined alendronate and calcitriol treatments improved bone loss owing to estrogen deficiency. On mechanical testing, although OVX significantly reduced bone strength of the femoral neck (by 29.3%) compared with the sham-control group, only the combined treatment significantly improved the fracture load at the femoral neck in OVX rats to the level of the sham-controls. The correlation of total BMC to fracture load was significant, but that of total BMD was not.</p> <p>Conclusion</p> <p>Our results showed that the combined treatment with alendronate and calcitriol significantly improved bone fragility of the femoral neck in OVX osteopenic rats.</p
Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1
CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action
A Universal Trend of Reduced mRNA Stability near the Translation-Initiation Site in Prokaryotes and Eukaryotes
Recent studies have suggested that the thermodynamic stability of mRNA secondary structure near the start codon can regulate translation efficiency in Escherichia coli, and that translation is more efficient the less stable the secondary structure. We survey the complete genomes of 340 species for signals of reduced mRNA secondary structure near the start codon. Our analysis includes bacteria, archaea, fungi, plants, insects, fishes, birds, and mammals. We find that nearly all species show evidence for reduced mRNA stability near the start codon. The reduction in stability generally increases with increasing genomic GC content. In prokaryotes, the reduction also increases with decreasing optimal growth temperature. Within genomes, there is variation in the stability among genes, and this variation correlates with gene GC content, codon bias, and gene expression level. For birds and mammals, however, we do not find a genome-wide trend of reduced mRNA stability near the start codon. Yet the most GC rich genes in these organisms do show such a signal. We conclude that reduced stability of the mRNA secondary structure near the start codon is a universal feature of all cellular life. We suggest that the origin of this reduction is selection for efficient recognition of the start codon by initiator-tRNA
A systematic review of patient reported factors associated with uptake and completion of cardiovascular lifestyle behaviour change
Background: Healthy lifestyles are an important facet of cardiovascular risk management. Unfortunately many individuals fail to engage with lifestyle change programmes. There are many factors that patients report as influencing their decisions about initiating lifestyle change. This is challenging for health care professionals who may lack the skills and time to address a broad range of barriers to lifestyle behaviour. Guidance on which factors to focus on during lifestyle consultations may assist healthcare professionals to hone their skills and knowledge leading to more productive patient interactions with ultimately better uptake of lifestyle behaviour change support. The aim of our study was to clarify which influences reported by patients predict uptake and completion of formal lifestyle change programmes. Methods: A systematic narrative review of quantitative observational studies reporting factors (influences) associated with uptake and completion of lifestyle behaviour change programmes. Quantitative observational studies involving patients at high risk of cardiovascular events were identified through electronic searching and screened against pre-defined selection criteria. Factors were extracted and organised into an existing qualitative framework. Results: 374 factors were extracted from 32 studies. Factors most consistently associated with uptake of lifestyle change related to support from family and friends, transport and other costs, and beliefs about the causes of illness and lifestyle change. Depression and anxiety also appear to influence uptake as well as completion. Many factors show inconsistent patterns with respect to uptake and completion of lifestyle change programmes. Conclusion: There are a small number of factors that consistently appear to influence uptake and completion of cardiovascular lifestyle behaviour change. These factors could be considered during patient consultations to promote a tailored approach to decision making about the most suitable type and level lifestyle behaviour change support
Strategies of Bacterial Over Expression of Membrane Transporters Relevant in Human Health: The Successful Case of the Three Members of OCTN Subfamily
A systematic review of the literature examining the diagnostic efficacy of measurement of fractionated plasma free metanephrines in the biochemical diagnosis of pheochromocytoma
BACKGROUND: Fractionated plasma metanephrine measurements are commonly used in biochemical testing in search of pheochromocytoma. METHODS: We aimed to critically appraise the diagnostic efficacy of fractionated plasma free metanephrine measurements in detecting pheochromocytoma. Nine electronic databases, meeting abstracts, and the Science Citation Index were searched and supplemented with previously unpublished data. Methodologic and reporting quality was independently assessed by two endocrinologists using a checklist developed by the Standards for Reporting of Diagnostic Studies Accuracy Group and data were independently abstracted. RESULTS: Limitations in methodologic quality were noted in all studies. In all subjects (including those with genetic predisposition): the sensitivities for detection of pheochromocytoma were 96%–100% (95% CI ranged from 82% to 100%), whereas the specificities were 85%–100% (95% CI ranged from 78% to 100%). Statistical heterogeneity was noted upon pooling positive likelihood ratios when those with predisposition to disease were included (p < 0.001). However, upon pooling the positive or negative likelihood ratios for patients with sporadic pheochromocytoma (n = 191) or those at risk for sporadic pheochromocytoma (n = 718), no statistical heterogeneity was noted (p = 0.4). For sporadic subjects, the pooled positive likelihood ratio was 5.77 (95% CI = 4.90, 6.81) and the pooled negative likelihood ratio was 0.02 (95% CI = 0.01, 0.07). CONCLUSION: Negative plasma fractionated free metanephrine measurements are effective in ruling out pheochromocytoma. However, a positive test result only moderately increases suspicion of disease, particularly when screening for sporadic pheochromocytoma
Codon bias at the 3 `-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli
The codon that follows the AUG initiation tripler (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgamo sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation. (C) 2001 Elsevier Science B.V. All rights reserved