Genomic analysis of the function of the transcription factor gata3 during development of the Mammalian inner ear

We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKB beta, a dramatic increase in Akt1/PKB alpha protein and relocation of Akt1/PKB alpha from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27(kip1), a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome

Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system

This work was supported by Medical Research Scotland (FRG353 to VAS), the FP7- Directorate-General for Research and Innovation of the European Commission (EU HEALTHF4-2012-305033 to Coordinating Action Systems Medicine to DJH); the Chief Scientist Office of Scotland (to DJH) and the Scottish Funding Council (to DJH and SPL).Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein levels. Yet studies into mRNA-protein correspondence have shown notoriously poor correlation between mRNA and protein expression levels, creating concern for inferences from only mRNA expression data. However, none of these studies have examined in particular differentially expressed mRNA. Here, we examined this question in an ovarian cancer xenograft model. We measured protein and mRNA expression for twenty-nine genes in four drug-treatment conditions and in untreated controls. We identified mRNAs differentially expressed between drug-treated xenografts and controls, then analysed mRNA-protein expression correlation across a five-point time-course within each of the four experimental conditions. We evaluated correlations between mRNAs and their protein products for mRNAs differentially expressed within an experimental condition compared to those that are not. We found that differentially expressed mRNAs correlate significantly better with their protein product than non-differentially expressed mRNAs. This result increases confidence for the use of differential mRNA expression for biological discovery in this system, as well as providing optimism for the usefulness of inferences from mRNA expression in general.Publisher PDFPeer reviewe

EUROSPINE 2017 FULL PAPER AWARD: Time to remove our rose-tinted spectacles: a candid appraisal of the relative success of surgery in over 4500 patients with degenerative disorders of the lumbar spine, hip or knee

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. Purpose: Studies comparing the outcome of spine surgery with that of large-joint replacement report equivocal findings. The patient-reported outcome measures (PROMs) used in such studies are typically generic and may not be sufficiently sensitive to the successes/failures of treatment. This study compared different indices of “success” in patients undergoing surgery for degenerative disorders of the lumbar spine, hip, or knee, using a validated, multidimensional, and joint-specific PROM. Methods: Preoperatively and 12 months postoperatively, 4594 patients (3937 lumbar spine, 368 hip, 269 knee) undergoing first-time surgery completed a PROM that included the Core Outcome Measures Index (COMI) for the affected joint. The latter comprises a set of single items on pain, function, symptom-specific well-being, quality of life, and disability—all in relation to the specified joint problem. Other single-item ratings of treatment success were made 12 months postoperatively. Results: In multiple regression analyses, controlling for confounders, the mean improvement in COMI at 12 months was greatest for the hip patients and lowest for those with degenerative spinal deformity (= the statistical reference group) (p < 0.05). Compared with spinal deformity, the odds of achieving “success” were: higher for hip (OR 4.6; 95% CI 2.5–8.5) and knee (OR 4.0; 95% CI 2.1–7.7) (no difference between spine subgroups) for “satisfaction with care”; higher for hip (OR 16.9; 95% CI 7.3–39.6), knee (OR 6.3; 95% CI 3.4–11.6), degenerative spondylolisthesis (OR 1.6; 95% CI 1.2–2.2), and herniated disc (OR 1.7; 95% CI 1.2–2.4) for “global treatment outcome”; and higher for hip (OR 13.8; 95% CI 8.8–21.6), knee (OR 5.3; 95% CI 3.6–7.8), degenerative spondylolisthesis (OR 1.6; 95% CI 1.3–2.1), and herniated disc (1.5; 95% CI 1.1–2.0) for “patient-acceptable symptom state”. Patient-rated complications were the greatest in degenerative spinal deformity (29%) and the lowest in hip (18%). Conclusions: The current study is the largest of its kind and the first to use a common, but joint-specific instrument to report patient-reported outcomes after surgery for degenerative disorders of the spine, hip, or knee. The findings provide a sobering account of the significantly poorer outcomes after spine surgery compared with large-joint replacement. Further work is required to hone the indications and patient selection criteria for spine surgery. The data should be used to lobby research funding-bodies, governmental agencies, industry, and charitable foundations to invest more in spine research/registries, in the hope of ultimately improving spine outcomes. Graphical abstract: These slides can be retrieved under Electronic Supplementary Material.[Figure not available: see fulltext.]

A quantitative estimation of the global translational activity in logarithmically growing yeast cells.

BACKGROUND: Translation of messenger mRNAs makes significant contributions to the control of gene expression in all eukaryotes. Because translational control often involves fractional changes in translational activity, good quantitative descriptions of translational activity will be required to achieve a comprehensive understanding of this aspect of biology. Data on translational activity are difficult to generate experimentally under physiological conditions, however, translational activity as a parameter is in principle accessible through published genome-wide datasets. RESULTS: An examination of the accuracy of genome-wide expression datasets generated for Saccharomyces cerevisiae shows that the available datasets suffer from large random errors within studies as well as systematic shifts in reported values between studies, which make predictions of translational activity at the level of individual genes relatively inaccurate. In contrast, predictions of cell-wide translational activity are possible from such datasets with higher accuracy, and current datasets predict a production rate of about 13,000 proteins per haploid cell per second under fast growth conditions. This prediction is shown to be consistent with independently derived kinetic information on nucleotide exchange reactions that occur during translation, and on the ribosomal content of yeast cells. CONCLUSIONS: This study highlights some of the limitations in published genome-wide expression datasets, but also demonstrates a novel use for such datasets in examining global properties of cells. The global translational activity of yeast cells predicted in this study is a useful benchmark against which biochemical data on individual translation factor activities can be interpreted

Artificial total disc replacement versus fusion for the cervical spine: a systematic review

Cervical total disc replacement (CTDR) has been increasingly used as an alternative to fusion surgery in patients with pain or neurological symptoms in the cervical spine who do not respond to non-surgical treatment. A systematic literature review has been conducted to evaluate whether CTDR is more efficacious and safer than fusion or non-surgical treatment. Published evidence up to date is summarised qualitatively according to the GRADE methodology. After 2 years of follow-up, studies demonstrated statistically significant non-inferiority of CTDR versus fusion with respect to the composite outcome ‘overall success’. Single patient relevant endpoints such as pain, disability or quality of life improved in both groups with no superiority of CTDR. Both technologies showed similar complication rates. No evidence is available for the comparison between CTDR and non-surgical treatment. In the long run improvement of health outcomes seems to be similar in CTDR and fusion, however, the study quality is often severely limited. After both interventions, many patients still face problems. A difficulty per se is the correct diagnosis and indication for surgical interventions in the cervical spine. CTDR is no better than fusion in alleviating symptoms related to disc degeneration in the cervical spine. In the context of limited resources, a net cost comparison may be sensible. So far, CTDR is not recommended for routine use. As many trials are ongoing, re-evaluation at a later date will be required. Future research needs to address the relative effectiveness between CTDR and conservative treatment

The eIF1A C-terminal domain promotes initiation complex assembly, scanning and AUG selection in vivo

Translation initiation factor 1A stimulates 40S-binding of the eukaryotic initiation factor 2 (eIF2)/GTP/Met-tRNA(i)(Met) ternary complex (TC) and promotes scanning in vitro. eIF1A contains an OB-fold present in bacterial IF1 plus N- and C-terminal extensions. Truncating the C-terminus (ΔC) or mutating OB-fold residues (66–70) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd(−) phenotype) in a manner suppressed by overexpressing TC. Consistent with this, both mutations diminished 40S-bound TC, eIF5 and eIF3 in vivo, and ΔC impaired TC recruitment in vitro. The assembly defects of the OB-fold mutation can be attributed to reduced 40S-binding of eIF1A, whereas ΔC impairs eIF1A function on the ribosome. A substitution in the C-terminal helix (98–101) also reduced 43S assembly in vivo. Rather than producing a Gcd(−) phenotype, however, 98–101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98–101 allows formation of aberrant 48S complexes in vitro and increases utilization of non-AUG codons in vivo. Thus, the OB-fold is crucial for ribosome-binding and the C-terminal domain of eIF1A has eukaryotic-specific functions in TC recruitment and scanning